Application of AR-V7 variable shearing sequence in resisting castration resistant tumors

An AR-V7 and sequence technology, applied in the field of biomedicine, can solve problems such as elevated PSA

Inactive Publication Date: 2018-11-23
天津市泌尿外科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the FDA-approved AR antagonist enzalutamide and androgen-suppressing abiraterone can bring survival benefits, there are still many CRPC patients who are insensitive to these two drugs from the beginning, and almost All treated patients eventually acquire drug resistance with elevated PSA, suggesting reactivation of the AR signaling pathway

Method used

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  • Application of AR-V7 variable shearing sequence in resisting castration resistant tumors
  • Application of AR-V7 variable shearing sequence in resisting castration resistant tumors
  • Application of AR-V7 variable shearing sequence in resisting castration resistant tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Construction and verification of positive castration-resistant prostate cancer cell line LNCaP-AI

[0085] 1. Androgen-free culture of cell lines

[0086] The androgen-dependent prostate cancer cell line LNCaP was cultured in RPMI-1640 medium with 10% activated charcoal androgen-depleted fetal bovine serum. Cells were cultured in a cell incubator at 37°C, 5% CO 2 .

[0087] 2. MTT method to detect cell proliferation activity

[0088] Count the cells to be tested and dilute to 2×10 4 / ml, inoculate in 96-well plate, add 100μl cell diluent to each well, about 2×10 3 Cells / well, 4-5 replicate wells were set for each group of experiments to reduce errors. Place at 37°C, 5% CO 2 Culture in the cell incubator, take out the 96-well plate 1, 2, and 3 days after inoculation, carefully suck off the cell culture medium and add 100 μl of 1×MTT solution to each well of the well to be tested, incubate for 4 hours in a 37°C incubator, and carefully suck out the upper Fo...

Embodiment 2

[0112] Example 2 The effect of CYTOR on the expression level of AR-V7 in castration-resistant prostate cancer cell lines

[0113] 1. Cell culture

[0114] LNCaP-AI and 22Rv1 cells were cultured in six-well plates in vitro, and cells were transfected 24 hours after plating.

[0115] 2. Transfection

[0116] 1) Design of shRNA

[0117] A short hairpin RNA targeting CYTOR (shCYTOR) and a control (shSCR) were designed and synthesized for the binding sequence of the long non-coding RNA CYTOR and AR-V7.

[0118] The shCYTOR sequence is as follows:

[0119] 5'-TCTATGTGTCTTAATCCCTTGTCCT-3' (SEQ ID NO.1)

[0120] Sequence of shSCR:

[0121] 5'-TTCTCCGAACGTGTCACGTTT-3' (SEQ ID NO.2)

[0122] 2) Lipofectamine transfection

[0123] Using Roche's Lipofectamine 2000 kit, the short hairpin RNA synthesized in vitro was mixed with liposome transfection reagent to transfect prostate cancer cells. For details, see the instructions.

[0124] 3. QPCR detection of CYTOR mRNA level in cells ...

Embodiment 3

[0142] Example 3 Bioinformatics prediction and evaluation of AR-V7 variable splicing functional sites

[0143] 1. Determination of the unique structure of AR-V7

[0144] 1) Use the gene module in the NCBI database to select the full-length transcript of the AR gene (androgen receptor[Homo sapiens(hTman)]), namely AR-FL (NM_000044.4), and the transcript 3, namely AR-V7 (NM_001348061.1 ) nucleic acid information;

[0145] 2) Use the Graphics module to compare the mRNA structures of AR-FL and AR-V7, and analyze the unique structure of AR-V7;

[0146] 3) Use the FASTA module to align the mRNA sequences, analyze the cleaved signal sequence, and determine the alternatively cleaved sequence of AR-V7.

[0147] 2. Analysis of alternative splicing functional sites

[0148] Use the HSF 3.1 module of the HTman Splicing Finder online prediction evaluation website, select Splice site analysis, enter the gene name AR, continue to select the transcripts identified in step 1, and use the ma...

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Abstract

The invention discloses application of an AR-V7 variable shearing sequence in resisting castration resistant tumors, and specifically the variable shearing sequence is as shown in SEQ ID NO.7. The invention also discloses CYTOR capable of controlling expression of the AR-V7, and discloses CYTOR which can be used for treating castration resistance, relapse and metastasis after hormone castration treatment of androgen receptor associated tumors. The invention also discloses antisense oligonucleotide capable of controlling AR-V7 expression, and provides a novel treatment way for castration resistance, relapse and metastasis after hormone castration treatment of androgen receptor associated tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the application of AR-V7 variable splicing sequence in resisting castration-resistant tumors. Background technique [0002] Prostate cancer is a common malignancy and the second leading cause of cancer death in men. In recent years, the incidence of prostate cancer in my country has shown a rapid upward trend year by year, from 5.8 / 100,000 in 2004 to 8 / 100,000 in 2009. The absolute value of the incidence rate increased the fastest in Hong Kong, China, from 16.5 / 100,000 in 1999 to 28.1 / 100,000 in 2010. In Taiwan and Shanghai, prostate cancer has ranked the fifth most common tumor in men and the first among urinary system tumors. Due to the relatively backward screening of prostate cancer susceptible population in my country, advanced prostate cancer accounts for about 70% of the first diagnosed prostate cancer; the proportion of newly diagnosed advanced prostate cancer in my country is m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/113A61K31/7088A61K45/00A61P35/00A61P35/04
CPCA61K31/7088A61K45/00A61P35/00A61P35/04C12N15/113C12N2310/11
Inventor 牛远杰尚芝群于健鹏冯睿
Owner 天津市泌尿外科研究所
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