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Nuclear localization signal sequences of ARID1B and application of nuclear localization signal sequences

A technology of nuclear localization signal and sequence, applied in the field of nuclear localization signal sequence

Pending Publication Date: 2021-01-22
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Designing inhibitors that selectively target ARID1B is highly challenging

Method used

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  • Nuclear localization signal sequences of ARID1B and application of nuclear localization signal sequences
  • Nuclear localization signal sequences of ARID1B and application of nuclear localization signal sequences
  • Nuclear localization signal sequences of ARID1B and application of nuclear localization signal sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] This example explores ARID1A in Hela, cervical cancer - / - Cell line C33a and ovarian clear cell carcinoma cell line ARID1A - / - Down-regulation of TNPO1 and ARID1B gene expression by siRNA interference in Tov-21g and the effects of TNPO1 and ARID1B on the proliferation of female reproductive system tumor cells, as follows:

[0052] 1. Cell level:

[0053] Construction of human cervical cancer and ovarian clear cell carcinoma cell lines stably knocking down the expression of TNPO1 and ARID1B: Since TNPO1 and ARID1B are highly expressed in cervical cancer tissues and ovarian clear cell carcinoma tissues, the expression of siRNA in cervical cancer and ovarian clear cell carcinoma cell lines Interfering methods down-regulate the expression of TNPO1 and ARID1B genes. Construct TNPO1 interference (shTNPO1-1 / 2 / 3) or overexpression (lentiTNPO1) lentiviral expression plasmids, transfect cervical cancer cell lines with the virus, and use puromycin to screen out cervical cancer c...

Embodiment 2

[0062] In this example, on the basis of Example 1, it is verified whether TNPO1 and ARID1B are combined with each other. Co-immunoprecipitation of TNPO1 and ARID1B proteins was carried out in Hela cells and C33a cervical cancer cells. The specific steps and results are as follows:

[0063] 1. Wash the cells twice with pre-cooled PBS, and dry the PBS for the last time;

[0064] 2. Add pre-cooled RIPA Buffer (1ml / 10 7 cells, 10cm dish or 150cm 2 Culture flask, 0.5ml / 5×10 6 Cells, 6cm dish, 75cm 2 culture bottle);

[0065] 3. Scrape the cells from the culture dish or flask with a pre-cooled cell scraper, transfer the suspension to a 1.5 EP tube, and shake slowly at 4°C for 15 minutes (Put the EP tube on ice and shake it horizontally bed);

[0066] Centrifuge at 14000g for 15min at 4.4°C and immediately transfer the supernatant to a new centrifuge tube

[0067] 5. Prepare Protein A agarose, wash the beads twice with PBS, and then prepare a 50% concentration with PBS. It is ...

Embodiment 3

[0080] This example verifies that the amino acid sequence is the nuclear localization signal sequence of SEQ ID No. 1-3, and the auxiliary nuclear import function of ARID1B, and each nuclear localization signal amino acid sequence is loaded on the pcDNA 3.1 GFP-GST tagged protein. The specific cell The steps and results of the slide immunofluorescence experiment are as follows:

[0081] 1. In the culture plate, soak the slide with the cells climbed in PBS 3 times, 3 minutes each time;

[0082] 2. Fix slides with 4% paraformaldehyde for 15 minutes, soak slides in PBS 3 times, 3 minutes each time;

[0083] 3. Permeabilize with 0.5% TritonX-100 (prepared in PBS) at room temperature for 20 minutes;

[0084] 4. Wash the slides with PBS for 3 times, each time for 3 minutes, blot the PBS with absorbent paper, add normal goat serum to the slides, and seal at room temperature for 30 minutes;

[0085] 5. Absorb the blocking solution with absorbent paper, without washing, add a suffici...

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Abstract

The invention provides nuclear localization signal sequences of ARID1B and application of the nuclear localization signal sequences. The nuclear localization signal sequences include one or more of amino acid sequences formed by combining TNPO1 and ARID1B and / or an amino acid sequence as shown in SEQ ID No.3. Experiments prove that the three nuclear localization signal sequences of the ARID1B provided by the invention can help the ARID1B enter a nucleus to play a role, so that the ARID1B can be used as an anti-tumor drug target, a small-molecule inhibitor of the ARID1B can cause synthetic lethality of ARID1A mutation, and a new research strategy is provided for preparation of drugs for treating cancers.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the nuclear localization signal sequence of ARID1B and its application. Background technique [0002] Gynecologic cancers, also known as cancers of the female reproductive tract, include malignant tumors from the cervix, fallopian tubes, ovaries, uterus, vulva, and vagina. Of these cancers, ovarian cancer was the most common cause of death, although endometrial cancer was the most common. Both ovarian and endometrial cancers are composed of several distinct histologic subtypes, each of which is often associated with distinct mutational backgrounds and features, such as BRCA mutations in high-grade serous carcinomas of the ovary . The latest genomic studies have shown that the genes encoding the subunits of the chromatin remodeling complex (SWI / SNF) are also highly mutated in gynecological tumors. ARID1A, a core subunit of SWI / SNF, is a tumor suppressor, and it is mutated in up to 57...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K45/06A61P15/00A61P35/00
CPCC07K14/47A61K45/06A61P35/00A61P15/00C07K2319/09
Inventor 杨毕康滕银成张志刚
Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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