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saRNA molecules of TPO genes and application of saRNA molecules

A molecular and genetic technology, applied in the field of molecular biology, can solve the problems of short residence time and limited clinical application value.

Active Publication Date: 2017-03-08
SHANGHAI SEVENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Another important factor affecting the curative effect of radioactive iodine is the residence time of iodine in the cell. If the residence time of radioactive iodine in the cell is short, its clinical application value will be limited to a certain extent.

Method used

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  • saRNA molecules of TPO genes and application of saRNA molecules
  • saRNA molecules of TPO genes and application of saRNA molecules
  • saRNA molecules of TPO genes and application of saRNA molecules

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: the preparation of saRNA

[0064] According to the RNAa principle summarized by Li et al. and the design software provided, saRNA was designed and synthesized for hTPO mRNA. The specific design steps are as follows:

[0065] 1. NCBI search hTPO gene characteristics are as follows:

[0066] (1) Human TPO is located on the chromosome: Chromosome 2: 1413461..1542727,

[0067] (2)Accession number:NG_011581

[0068] (3) Length: 129267bp

[0069](4) UCSC retrieved the sequence of 1000 genes upstream 6000bp downstream of TSS as shown in SEQ ID NO: 1, wherein the 6001st to 6010th bases in the sequence SEQ ID NO: 1 are the transcription initiation point TSS.

[0070] According to the principle of saRNA design, a total of 6 saRNA sequences were designed and synthesized within the range of 6000bp upstream and 1000bp downstream of the TSS [saRNA-758(sa-758), saRNA-3073(sa-3073), saRNA-3860(sa-3860), saRNA-4576 (sa-4576), saRNA-5198 (sa-5198), saRNA-5640 (sa-5640)]...

Embodiment 2

[0074] Example 2 Effect of saRNA on hTPO expression

[0075] Liver cancer cells HepG2 were treated with DMEM containing 10% fetal bovine serum, 5% CO 2 , 37 ℃ incubator culture. Cells in logarithmic growth phase, 2×10 5 Inoculated in six-well plates and cultured overnight. On the next day, dsRNAs and dscontrol were transfected into liver cancer cells HepG2 by liposome Lipofectamine2000 at a final concentration of 50nM. 72 hours after transfection, the expression of hTPO mRNA in each group was semi-quantitatively analyzed by RT-PCR, and the up-regulation of HTPO expression by RNAa was analyzed. degree.

[0076] Western blot analysis

[0077] The cells were washed with PBS buffer, lysed with RIPA protein lysate on ice for several minutes, and then centrifuged at 12,000 rpm / min for 15 minutes to collect the supernatant; the protein concentration was detected by BCA method. After 10% SDS-PAGE gel electrophoresis for about 2 hours, the protein was transferred to PVDF membrane ...

Embodiment 3

[0079] Embodiment 3 Radionuclide uptake experiment

[0080] After transfecting two HepG2 cells with hNIS-saRNA and / or hTPO-saRNA (saRNA-3073) sequences, add Na 125 I solution, after continuing to incubate for 2 hours, measure the radioactive counts in the cells of each group at different time points. The results show( Figure 4 ), the intracellular radioactive count of the combined transfection group was higher than that of the hNIS-saRNA transfected cells alone (P<0.01), and at 30 min, the intracellular radioactive count of the combined transfection group was 3.03 times that of the hNIS-saRNA transfected group.

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Abstract

The invention relates to the field of molecular biology medicine. saRNA molecules of TPO genes are synthesized, the sequence of the saRNA molecules and the sequence SEQ ID NO:1 of the upstream 6,000bp and downstream 1,000bp areas of a TPO gene transcriptional start site are complementary, and low-expression or non-expression TPO genes in tumor cells can be effectively activated. A biological effect of promoting the tumor cells to hold radionuclide 131I is achieved, a novel method and novel molecular targets are provided for combining the gene technology with the radionuclide therapy for targeted therapy of tumors.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to the saRNA molecule of TPO gene and its application. Background technique [0002] Gene therapy is a new treatment method that introduces normal genes or therapeutic genes into target cells to achieve the purpose of disease treatment. In recent years, many studies on gene therapy methods for tumors have been carried out all over the world. Among them, RNA activation (RNA activation, RNAa) is a new gene technology discovered and developed in recent years. The expression technology can activate the endogenous gene of the cell, so as to realize the new technology of up-regulating the expression of the target gene. The development of RNA activation technology provides an extremely promising option for combining gene technology with radionuclide therapy for targeted treatment of tumors. [0003] Sodium iodide symporter (Human sodium iodide symporter, hNIS) and human thyroid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85A61K51/04A61P35/00A61K101/02
CPCA61K51/0491C12N15/113C12N2310/10
Inventor 夏伟叶颖庄菊花王国玉
Owner SHANGHAI SEVENTH PEOPLES HOSPITAL
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