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Application of TMUB1 protein in preparation of tumor immunosuppressive molecular detection agent

An immunosuppressive molecule and inhibitor technology, which is applied in the application field of TMUB1 protein in the preparation of tumor immunosuppressive molecule detection agent, can solve the problems of poor immunotherapy effect and poor prognosis of tumor treatment, and achieve the effect of hindering immune escape.

Active Publication Date: 2021-03-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the high expression level of TMUB1 in tumor tissue can predict the high level of PD-L1, which indicates the poor effect of immunotherapy in tumor tissue and the poor prognosis of tumor treatment.

Method used

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  • Application of TMUB1 protein in preparation of tumor immunosuppressive molecular detection agent
  • Application of TMUB1 protein in preparation of tumor immunosuppressive molecular detection agent
  • Application of TMUB1 protein in preparation of tumor immunosuppressive molecular detection agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of TMUB1 small molecule RNA inhibitor

[0035]According to the TMUB1 gene nucleotide sequence (Gene ID: 83590, http: / / sidirect2.rnai.jp / ), according to the principle of complementary base pairing and the principle of the lowest energy, two cholesterol-modified small RNA nucleotide sequences were obtained, They are TMUB1 sequence 1 and TMUB1 sequence 2, which are TMUB1 small molecule RNA inhibitors.

[0036] TMUB1 sequence 1: 5'-GGCUGAAAUUCCUCAAUGA-3',

[0037] TMUB1 sequence 2: 5'-CUACGGCUGAAAUUCCUCA-3'.

[0038] At the same time, according to NCBI BLAST, random sequences (nonsense targeting) were designed as the control group for cholesterol-modified small RNAs: AAC ACG UCU AUACGC.

[0039] TMUB1 molecular detection primers (TMUB1 F, TMUB1 R) and internal reference primers (GAPDHF, GAPDH R) were designed according to NCBI BLAST:

[0040] TMUB1 F: 5'-AGGTGACCGTCCTTTTCTCG-3',

[0041] TMUB1R: 5'-ATTTCAGCCGTAGCACGAGG-3';

[0042] GAPDH F...

Embodiment 2

[0044] Embodiment 2, suppress the mRNA expression of TMUB1 in breast cancer cell line

[0045] Using the TMUB1 sequence 1, TMUB1 sequence 2 and nonsense targeting control sequence prepared in Example 1, they were transfected into the human breast cancer cell line MDA-MB-231 (purchased from American Standard Cell Culture Center, ATCC), respectively constructed two TMUB1-specific knockdown cell lines, denoted as targeted knockdown #1 and targeted knockdown #2, and a cell line with a nonsense targeting sequence , recorded as nonsense targeted knockdown control; use DMEM medium containing 10% FBS to continue at 37°C, 5% CO 2 Cells were harvested after 36 hours of culture. Under the same conditions, the human breast cancer cell line MDA-MB-231 was replaced with the human breast cancer cell line MDA-MB-468 (purchased from American Standard Cell Culture Center, ATCC).

[0046] Extract RNA according to the instructions of TRIzol kit (Ambion); use NanoDrop (Thermo) to quantify RNA ac...

Embodiment 3

[0047] Example 3. Targeted knockdown of TMUB1 inhibits the accumulation of PD-L1 protein in breast cancer cells

[0048] Human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were purchased from American Standard Cell Culture Center (ATCC), using DMEM / F12 medium (Gibco) containing 10% FBS (Bovogen), 37°C, 5% CO 2 Cultured in an incubator, the MycoAlert kit (Lonza) detects that the cells have no mycoplasma contamination and no cross-contamination.

[0049] Using the TMUB1 small molecule RNA inhibitor prepared in Example 1, carry out plasmid transfection according to the instruction manual of Lipofectamine 2000 (ThermoScientific) reagent, specifically: according to the method of ViraPowerTMLentiviralExpression System (Thermo Scientific), the TMUB1 sequence 1 in Example 1 was respectively , TMUB1 sequence 2 and nonsense targeting sequence were transferred into the human breast cancer cell line MDA-MB-231, and two TMUB1-specific knockdown cell lines were respectively construct...

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Abstract

The invention discloses an application of a TMUB1 protein in preparation of a tumor immunosuppressive molecule detection agent, and provides a method for detecting the expression level of the TMUB1 protein and an application of a TMUB1 protein inhibitor in inhibition of tumor immunosuppressive molecules at the same time, in the invention, the TMUB1 with low expression level can significantly reduce the protein level of PD-L1, PD-L1 protein accumulation in tumor cells is blocked from the root of PD-L1 protein synthesis, and immune escape of the tumor cells is blocked.

Description

[0001] (1) Technical field [0002] The invention relates to the application of a TMUB1 protein in the preparation of a molecular detection agent for tumor immunosuppression. [0003] (2) Background technology [0004] According to the latest World Cancer Report 2020 from the World Health Organization (WHO) in February, the number of cancer cases worldwide could increase by 60% in the next two decades. In low- and middle-income countries, the increase could be as high as 81%. One-sixth of the world dies of cancer every year, and the cancer burden is increasing. In 2018, approximately 9.6 million people worldwide died of cancer. The experience of global "cancer prevention and treatment" shows that it is of great significance to implement comprehensive strategies such as tobacco control, vaccination, regular cancer screening, and effective diagnosis and treatment techniques. In addition to traditional surgical resection, radiotherapy, and chemotherapy, cancer treatment methods...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/574C12Q1/6886A61K45/00A61K31/7088A61P35/00
CPCG01N33/6893G01N33/57484C12Q1/6886A61K45/00A61K31/7088A61P35/00C12Q2600/118C12Q2600/158
Inventor 林爱福瞿蕾吴敏洁
Owner ZHEJIANG UNIV
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