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Application of small activating RNA (Ribonucleic Acid) of INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of prostate cancer fighting medicament

An INTS6, prostate cancer technology, applied in gene therapy, drug combination, anti-tumor drugs, etc., can solve the problem of expression loss, and achieve the effect of exact activation, low cost and safe application

Inactive Publication Date: 2012-09-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Expression loss of INTS6 in prostate cancer tissues and cell lines

Method used

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  • Application of small activating RNA (Ribonucleic Acid) of INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of prostate cancer fighting medicament
  • Application of small activating RNA (Ribonucleic Acid) of INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of prostate cancer fighting medicament
  • Application of small activating RNA (Ribonucleic Acid) of INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of prostate cancer fighting medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 dsRNAs up-regulate the expression of INTS6 gene in prostate cancer cells

[0022] Experimental program:

[0023] 1. Cell lines: human hormone-independent prostate cancer cell lines PC3, Du145 (Shanghai Cell Institute)

[0024] 2. Synthesis of dsRNAs: dsRNAs were chemically synthesized by Shanghai Jikai Biotechnology Co., Ltd.

[0025] 3. Experimental group:

[0026] 20nM dsINTS6-374 and -390 groups, nonsense dsRNA control group (dsControl) and blank transfection reagent group (mock). dsControl group dsRNA is non-homologous to known human genome sequence: sense strand, 5'-ACU ACU GAG UGA CAG UAG A[dTdT]-3' (SEQ ID NO:5); antisense strand, 5'-UCU ACU GUC ACU CAG UAG U[dTdT]-3' (SEQ ID NO: 6).

[0027] 4. Cell culture and transfection:

[0028] The cells were cultured in RPMI1640 medium containing 10% inactivated newborn bovine serum, placed in a 37oC cell culture box with a CO2 content of 5%. The day before transfection, the cells were subcultured and ...

Embodiment 2

[0038] Example 2 Inhibitory effect of dsINTS6-374 / -390 up-regulating INTS6 on hormone-independent prostate cancer cells in vitro

[0039] Experimental program:

[0040] 1. Cell line: same as Example 1.

[0041] 2. dsRNAs synthesis: with embodiment 1.

[0042] 3. Experimental grouping: with embodiment 1.

[0043] 4. Cell culture and transfection: the same as in Example 1.

[0044] 5. Tetrazolium salt (MTT) colorimetric method:

[0045] Take the cancer cells in the logarithmic growth phase, trypsinize to make a single cell suspension, and plant the cells in a 96-well culture plate at a density of 2000-5000 cells per well (to ensure the accuracy of the results, the The cell attachment rate, doubling time and the growth curve of cells with different inoculated numbers, and then determine the inoculated number of cells in each well to ensure that the cells will not be overfilled when the culture is terminated), and the culture plate is placed at 37 o C, 5% CO 2 After culturin...

Embodiment 3

[0061] Example 3 dsINTS6-374 up-regulates INTS6 to inhibit the growth of hormone-independent prostate cancer in vivo

[0062] Experimental program:

[0063] 1. Cell line: human hormone-independent prostate cancer cell line PC3.

[0064] 2. Animal model: 4-week-old BALB-c nude mice were used to bear tumors subcutaneously. After tumor formation, they were divided into non-sense dsRNA control group and dsINTS6-374 / -390 RNA group for intervention, with 4 rats in each group.

[0065] 3. Dosing regimen: intratumoral injection of 30 μg of liposome-coated RNA, once every 3 days, for a total of three weeks.

[0066] 4. Observation of results: Synchronous observation of tumor size changes. Finally, nude mice were sacrificed, and the expression of INTS6 protein in tumor tissues was observed by immunohistochemical staining.

[0067] The result is as follows:

[0068] Small activating RNA can activate the expression of INTS6 in subcutaneous xenografts of PC3 nude mice, and at the same...

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Abstract

The invention provides application of small RNA (Ribonucleic Acid) of a targeted up-regulation INTS6 (Homo Sapiens Integrator Complex Subunit 6) gene to preparation of a hormone refractory prostate cancer fighting medicament. A nucleotide sequence of the small RNA consists of four pieces of positive-sense strands and anti-sense strands of 21 nucleotides; two nucleotides, which are generally dTdT, are overhung at the end 3' of each strand; and the middle 19 nucleotides are paired. According to the application, the expression of INTS6 gene in a hormone refractory prostate cancer cell is induced by the small double-stranded RNA of a specific sequence, so that the proliferation capacity and the movement capacity of tumor cells are inhibited, and the aim of inhibiting tumor growth and development is fulfilled. The small RNA is easy to operate, low in cost and less in using amount, and a good activation effect can be achieved with 20 nM transfection concentration; and the activation effect is clear, and the target gene mRNA (micro Ribonucleic Acid) level and the protein level are both raised. In addition, the saRNA (small activating RNA) induced gene expression belongs to apparent genetic regulation, the integrity of a genome is not damaged, and thus the application is safe.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of small activating RNA targeting up-regulation of INTS6 gene in the preparation of anti-prostate cancer drugs, especially hormone-independent prostate cancer drugs. Background technique [0002] Prostate cancer is seriously threatening the health of men over 50 years old in our country. Endocrine therapy is a commonly used method in the clinical treatment of prostate cancer. However, the vast majority of prostate cancer patients progress within 1 to 2 years of endocrine therapy, and continuing to use endocrine therapy drugs cannot control the progression of the disease. The transformation of prostate cancer cells from hormone-dependent to hormone-independent is the main reason for the failure of endocrine therapy and the death of patients with prostate cancer. [0003] In 2006, Li et al. reported that dsRNA (double-stranded RNA, double-stranded RNA) targeting the gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00
Inventor 谢立平陈弘林奕伟郑祥毅毛祺琦杨凯秦杰茅叶青胡政麾金勇丰
Owner ZHEJIANG UNIV
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