Single-chain antisense RNA expressed by targeted silent H19RNA and method for detecting effect thereof

Abstract

The invention belongs to the field of molecular biology, and discloses a single-stranded antisense RNA expressed by silent non-coding oncogene H19RNA and a method for detecting the effect thereof. The single-stranded antisense RNA is a 23nt, 27nt, 31nt, 35nt and 39nt single-stranded antisense RNA designed and synthesized by taking partial sequences of the fifth exon of the H19RNA as target sites, wherein 5' and 3' are both hydroxyl without other chemical modification. The sasRNA can durably, efficiently and specifically inhibit the expression of the H19RNA by utilizing a liposome mediated method to transfect human cervical cancer Hela cells and human prostate cancer PC3 cells. The reduction of the expression level of the H19RNA mediated by the flow cell testing sasRNA leads the cell cycleto be arrested in G2 / M phase. The single-strand antisense RNA of the invention has the advantages of obvious effect, simple and convenient operation of the detection method, low cost and wide application prospect.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a single-stranded antisense RNA for silencing the expression of non-coding cancer gene H19 RNA and a method for detecting its effect. Background technique [0002] Gene silencing refers to the biological phenomenon that a specific gene in an organism cannot be expressed or its expression is reduced due to the existence of various regulatory factors. It is not only an important way for an organism to regulate gene expression and maintain normal physiological activities, but also an Functional research and annotation, an important way to discover drug targets for disease treatment and even implement disease gene therapy. Currently commonly used gene silencing techniques mainly include three categories, the first category is antisense DNA (antisense oligonucleotide, ASO) technology. Antisense DNA refers to an antisense oligonucleotide that can bind to the mRNA of a specific gene in t...

AI Technical Summary

Problems solved by technology

These three methods have their own advantages and disadvantages, but in practical applications, they often face the same problem: (1) the efficiency of gene silencing is not high, resulting in the expression of the target gene cannot be effectively inhibited; (2) the duration of gene silencing Short, resulting in rapid recovery of the expression of the target gene, it is difficult to complete functional analysis; (3) the specificity of gene silencing is not high, causing off-target effects or immune side effects, resulting in misjudgment of the results (Lin, X., Ruan, X., Anderson, M.G., McDowell, J.A., Kroeger, P.E., Fesik, S.W., and Shen, Y. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 2005, 33, 4527-4535 ; Tschuch, C., Schulz, A., Pscherer, A., Werft, W., Benner, A., Hotz-Wagenblatt, A., Barrionuevo, L.S., Lichter, P., and Mertens, D. Off-target effects of siRNA specific for GFP. BMC Mol Biol 2008, 9, 60; Kleinman, M.E., Yamada, K., Takeda, A., Chandrasekaran, V., Nozaki, M., Baffi, J.Z., Albuquerque, R.J., Yamasaki, S ., Itaya, M., Pan, Y. Sequence-and target-independent angiogenesis suppression by siRNA via TLR3. Nature 2008, 452, 591-597.)

In addition, for the mRNA of a specific target gene, the local secondary structure and other characteristics make different sites show different accessibility (accessibility), resulting in huge differences in gene silencing effects

Therefore, finding effective gene silencing methods and effective target gene action sites is still a huge challenge in practical research work

Images