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SiRNA molecule against human cysteine proteinase inhibitor B (CSTB) and application thereof

A cysteine ​​protease and inhibitor technology, applied in the field of tumor molecular biology, can solve the problems of no CSTB, general knockdown effect, no knockdown effect, etc., to reduce CSTB mRNA and protein expression, knockdown significant effect

Pending Publication Date: 2019-07-26
JINSHAN HOSPITAL FUDAN UNIV
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Problems solved by technology

However, in this literature, CSTB has an inhibitory effect on gastric cancer, and the knockdown effect of si-CSTB on CSTB in this paper is average.
[0005] In summary, there are no reports of CSTB in the treatment of ovarian cancer, and there are no siRNA molecules that have a significant knockdown effect against CSTB and have promising drug development prospects.

Method used

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  • SiRNA molecule against human cysteine proteinase inhibitor B (CSTB) and application thereof
  • SiRNA molecule against human cysteine proteinase inhibitor B (CSTB) and application thereof
  • SiRNA molecule against human cysteine proteinase inhibitor B (CSTB) and application thereof

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Embodiment 1

[0032] 1. Experimental method

[0033] ① Construction of CSTB-shRNA lentiviral vector

[0034] The lentiviral vector pLKO.1-TRC cloning vector (addgene product number 10878) was used to construct shRNA lentivirus. The main steps are as follows:

[0035] a) double-digest the pLKO.1-TRC vector using AgeI and EcoRI restriction endonucleases;

[0036] b) Synthesize CSTB-shRNA and NC-shRNA sequences for annealing, the sequences are as follows:

[0037] CSTB-shRNA vector insert sequence:

[0038] sense 5'-ccggGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCCTTTTTTg-3' (SEQ ID NO: 1),

[0039] antisense 5'-aattcAAAAAAGGACAAACTACTTCATCAACTCGAGTTGATGAAGTAGTTTGTCC-3' (SEQ ID NO: 2);

[0040] The insert sequence of NC-shRNA vector is:

[0041] sense 5'-ccggTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTg-3' (SEQ ID NO: 3),

[0042] antisense 5'-aattcAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3' (SEQ ID NO: 4).

[0043] c) Insert the annealed sequence into the linearized pLKO...

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Abstract

The invention relates to an siRNA molecule against human cysteine proteinase inhibitor B (CSTB) and application thereof. An siRNA having a remarkable knockdown effect and a DNA double-stranded sequence expressing shRNA are designed against the human CSTB, and a lentiviral vector comprising the double-stranded DNA sequence is constructed. By biological function experiments of ovarian cancer cells,it is proved that the siRNA and an shRNA-expressing construct can significantly reduce the expression of CSTB-mRNA and protein and cause proliferation inhibition of ovarian cancer cells, so that the cells are retarded in a G2 / M phase, apoptosis of the ovarian cancer cells is promoted, and thus the siRNA and an shRNA-expressing construct can be developed as a small molecule therapeutic drug, or research and development reagents for studying a pathological mechanism of ovarian cancer.

Description

technical field [0001] The invention relates to the technical field of tumor molecular biology, in particular to a siRNA molecule targeting human cysteine ​​protease inhibitor B and its application. Background technique [0002] Ovarian cancer is the eighth most common cancer among women worldwide, but its mortality rate is the highest among gynecological cancers. The main reason is that the early symptoms of ovarian cancer are not obvious, and once clinically diagnosed, most of them are in the advanced stage and the prognosis is poor. Finding biomarkers that can be used to diagnose and treat ovarian cancer is very necessary. [0003] The applicant's previous work has found that cysteine ​​protease inhibitor B (Cystatin B, CSTB) is a marker of human ovarian cancer (Ovarian Cancer, OC), and found that transforming growth factor (TGF-β) 1 can regulate CSTB expression (Int J Oncol 2014,44:1099). However, the function of CSTB in OC and the specific mechanism of its regulation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C07K14/155C12N5/10
CPCC12N15/113C12N15/86C07K14/005C12N5/0693C12N2740/15043C12N2740/15023C12N2510/00
Inventor 许国雄管文彩汪星星
Owner JINSHAN HOSPITAL FUDAN UNIV
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