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 lack of tumor cell apoptosis, single target, unsatisfactory treatment effect, etc., to promote tumor cell apoptosis, improve effect, improve The effect of recognition

Inactive Publication Date: 2011-02-16
NANJING UNIVERSTIY SUZHOU HIGH TECH INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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
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Example Embodiment

[0031] Example 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 sequence of human and mouse TGF-β mRNA in the PUBMED gene library, using siRNA design software (Dharmacon RNAi Technologies), several corresponding matching short antisense sequences were screened, each containing 19 ribonucleosides. The corresponding siRNA (Metabion, Germany) was synthesized in vitro, and two free uridines were linked to the 3'end of each single strand, which was used as the OH-TGF-β group for in vitro screening and tumor suppression experiments.

[0034] The synthesized siRNA sequence (OH-TGF-β) was mixed and coated with liposomes (Lipofectamine 2000) in OptiMEM medium at 0.5 μg / μl in vitro, and then transfected into pancreatic cancer cells in vitro for 24h at different times Cell RNA was extracted at 48h and 72h, and the mRNA expression of TGF-β1 was detected by quanti...

Example Embodiment

[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), PaTu8988t (DSMZ, German Microbial Culture Collection), BxPC-3 (Cell Resources of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) Center), IMIM PC-1 (presented by Dr. Patrick Michl of Marburg University, Germany, and its isolation and in vitro culture methods are described in detail by ViláMR et al. [1] . The Panc02 cell line is a mouse pancreatic adenocarcinoma induced by methylcholanthrene (University of Munich, Germany), and its induction and culture methods were first disclosed by Corbett et al. [2] .

[0051] 2.1 In vitro transfection: tumor cell line 3x10 5 Each well is planted in a 6-well cell culture plate. The RNA was coated with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and then dissolved in OptiMEM m...

Example Embodiment

[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, the cell culture supernatant, mouse serum or tumor entity homogenate supernatant was collected at different time points , Use enzyme-linked immunosorbent assay kit to detect IFN-α (PBL Interferon source), IP-10 (R&D Systems), TGF-β (eBiosciences) and TNF-α (BD Biosciences) according to the method provided by the manufacturer's instructions. The results showed that: ppp-TGF-β transfected mouse pancreatic cancer cell Panc02 in vitro, the TGF-β protein level was significantly reduced after 24 hours (see figure 2 A), the level of interferon-induced protein is significantly higher than that of OH-TGF-β (see figure 2 B); The plasma TGF-β levels of tumor-bearing mice treated with mu...

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