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CYTOR and application of inhibitor thereof to resistance to castration resistant tumor

An inhibitor, tumor technology, applied in the field of biomedicine, can solve problems such as PSA increase

Active Publication Date: 2019-05-14
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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  • CYTOR and application of inhibitor thereof to resistance to castration resistant tumor
  • CYTOR and application of inhibitor thereof to resistance to castration resistant tumor
  • CYTOR and application of inhibitor thereof to resistance to castration resistant tumor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Construction and verification of positive castration-resistant prostate cancer cell line LNCaP-AI

[0081] 1. Androgen-free culture of cell lines

[0082] 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 .

[0083] 2. MTT method to detect cell proliferation activity

[0084] 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 at 37°C for 4 hours, and carefully suck out the upper For the supe...

Embodiment 2

[0108] Example 2 The effect of CYTOR on the expression level of AR-V7 in castration-resistant prostate cancer cell lines

[0109] 1. Cell culture

[0110] LNCaP-AI and 22Rv1 cells were cultured in six-well plates in vitro, and cells were transfected 24 hours after plating.

[0111] 2. Transfection

[0112] 1) Design of shRNA

[0113] 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.

[0114] The shCYTOR sequence is as follows:

[0115] 5'-TCTATGTGTCTTAATCCCTTGTCCT-3' (SEQ ID NO.1)

[0116] Sequence of shSCR:

[0117] 5'-TTCTCCGAACGTGTCACGTTT-3' (SEQ ID NO.2)

[0118] 2) Lipofectamine transfection

[0119] 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.

[0120] 3. QPCR detection of CYTOR mRNA level in cells ...

Embodiment 3

[0138] Example 3 Bioinformatics prediction and evaluation of AR-V7 variable splicing functional sites

[0139] 1. Determination of the unique structure of AR-V7

[0140] 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;

[0141] 2) Use the Graphics module to compare the mRNA structures of AR-FL and AR-V7, and analyze the unique structure of AR-V7;

[0142] 3) Use the FASTA module to align the mRNA sequences, analyze the cleaved signal sequence, and determine the alternatively cleaved sequence of AR-V7.

[0143] 2. Analysis of alternative splicing functional sites

[0144]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 max...

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PUM

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Abstract

The invention discloses CYTO. CYTO can regulate and control AR-V7 expression, and there is a hint that CYTRO can be used for treating castration resistance and relapse and metastasis after an androgenreceptor related tumor receives hormone castration treatment. The invention further discloses antisense oligonucleotide capable of regulating and controlling AR-V7 expression. A new treatment purposeis provided for tumor treatment and especially castration resistance and relapse and metastasis after the androgen receptor related tumor receives hormone castration treatment.

Description

[0001] This application is a divisional application for the application number 2018107698084, the application date is July 13, 2018, and the invention title is "Application of AR-V7 variable splicing sequence in castration-resistant tumors". technical field [0002] The invention belongs to the field of biomedicine and relates to the application of CYTOR and its inhibitors in castration-resistant tumors. Background technique [0003] 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 screen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61K45/00A61P35/00A61P35/04
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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