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TGF-beta specific siRNA containing free triphosphoric acid group and application thereof

A triphosphate group, TGF- technology, applied in the field of RNA interference, can solve the problems of single target, lack of tumor cell apoptosis, and unsatisfactory treatment effect, so as to improve recognition, improve effect, and promote tumor cell apoptosis death effect

Inactive Publication Date: 2013-04-03
NANJING UNIVERSTIY SUZHOU HIGH TECH INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of clinical tumor treatment in the early stage is not ideal, and the reason may be related to its single target, inability to effectively activate the body's immune surveillance / killing function against tumors, and lack of direct promotion of tumor cell apoptosis.

Method used

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  • TGF-beta specific siRNA containing free triphosphoric acid group and application thereof
  • TGF-beta specific siRNA containing free triphosphoric acid group and application thereof
  • TGF-beta specific siRNA containing free triphosphoric acid group and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Synthesis of ppp-TGF-β of the present invention

[0032] 1.1 Design and screening of human and mouse TGF-β specific small interfering RNA:

[0033] According to the complete coding sequences of human and mouse TGF-βmRNA in the PUBMED gene bank, using siRNA design software (Dharmacon RNAi Technologies), several matching antisense short sequences were screened out, each containing 19 ribonucleosides. The corresponding siRNA (Metabion, Germany) was synthesized in vitro, and two free uridine nucleosides were linked to the 3' end of each single strand, and used as the OH-TGF-β group for in vitro screening and tumor inhibition experiments.

[0034] The synthetic siRNA sequence (OH-TGF-β) was mixed and coated with liposome (Lipofectamine 2000) in OptiMEM medium at 0.5 μg / μl in vitro, and transfected into pancreatic cancer cells in vitro, at different times for 24 hours , 48h and 72h to extract cellular RNA, use quantitative RT-PCR to detect the mRNA expression of...

Embodiment 2

[0049] Example 2 Quantitative RT-PCR detection of ppp-TGF-β gene silencing and immune induction function:

[0050] Transfection of cells and mice: human pancreatic cancer cell lines PANC-1, MIAPaCa-2 (from ATCC, USA), PaTu 8988t (DSMZ, German Culture Collection of Microorganisms), BxPC-3 (cells of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) Resource Center), IIMIM PC-1 (gifted by Dr. Patrick Michl, University of Marburg, Germany, and its isolation and in vitro culture methods are described in detail by ViláMR et al. [1] . Panc02 cell line is a mouse pancreatic adenocarcinoma induced by methylcholanthracene (Germany University of Munich), and its induction and culture methods are first disclosed by Corbett et al. [2] .

[0051] 2.1 In vitro transfection: tumor cell line by 3 × 10 5 Each well was seeded in a 6-well cell culture plate. RNA was coated with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and then dissolv...

Embodiment 3

[0055] Example 3 Detection of ppp-TGF-β gene silencing and immune induction function by enzyme-linked immunosorbent assay (ELISA)

[0056] After mouse pancreatic cancer tumor cells Panc02 or experimental mice were transfected with RNA or received RNA treatment according to the method described in Example 2, cell culture supernatant, serum of mice or homogenate supernatant of tumor entities were collected at different time points , IFN-α (PBL Interferon source), IP-10 (R&D Systems), TGF-β (eBiosciences) and TNF-α (BD Biosciences) were detected with enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions. The results showed that: ppp-TGF-β transfected mouse pancreatic cancer cell Panc02 in vitro, TGF-β protein level significantly decreased after 24 hours (see figure 2 A), the level of interferon-induced protein was significantly higher than that of OH-TGF-β (see figure 2 B); the tumor-bearing mice treated with mouse ppp-TGF-β, its plasma TGF-β leve...

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Abstract

The invention belongs to the technical field of RNA interference, and discloses TGF-beta specific siRNA containing a free triphosphoric acid group and application thereof. The 5' ends of antisense strand and sense strand sequences of the TGF-beta specific siRNA containing the free triphosphoric acid group are guanosine, and the free triphosphoric acid group is modified on the pentose 3' site of the guanosine. By combining the TGF-beta specific gene silencing mechanism and the antiviral inherent immune mechanism of an induced eukaryotic cell, the siRNA can inhibit important molecule TGF-beta of mediated tumor immunologic escape, effectively activate the anti-tumor immunity of the body and remarkably improve the effect of treating tumor. The siRNA effectively solves the problem that the traditional siRNA (OH-RNA) only has single gene silencing function but poor tumor treatment effect. The ppp-TGF-beta can be applied to preparing a medicament for treating the tumor, in particular pancreatic cancer.

Description

technical field [0001] The invention belongs to the technical field of RNA interference and relates to siRNA and its application, in particular to TGF-beta specific siRNA containing free triphosphate groups and its application. Background technique [0002] Due to early metastasis, high resistance to chemotherapy and radiotherapy, the prognosis of pancreatic cancer is extremely poor. Abnormal expression of growth transforming factor β (TGF-β) plays a key role in the progression of pancreatic cancer. Highly expressed TGF-β can promote tumor growth, invasion, metastasis and angiogenesis. In addition, tumor-derived TGF-β has a strong immune-suppressing effect and can help tumors establish immune escape mechanisms. How to suppress tumor-mediated immunosuppression is a major challenge in the future treatment of pancreatic cancer. For this important molecule related to tumor growth, invasion, metastasis and immune escape, one of the most effective methods is to use small interfe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00C12N15/113A61P35/00A61P1/18
Inventor 魏继武马柯思·斯诺
Owner NANJING UNIVERSTIY SUZHOU HIGH TECH INST
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