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Application of reagents for inhibiting sgce gene

An inhibitor, gene technology, applied in the biological field

Active Publication Date: 2021-03-30
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first purpose of the present invention is to solve the problem of chemotherapy resistance in patients in the prior art, and provide the application of shRNA, siRNA and SGCE monoclonal antibody that inhibit the SGCE gene

Method used

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  • Application of reagents for inhibiting sgce gene
  • Application of reagents for inhibiting sgce gene
  • Application of reagents for inhibiting sgce gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Low expression of SGCE makes TNBC cell lines more sensitive to chemotherapeutic drugs

[0027] (1) Analyze the relationship between the expression of SGCE and the survival curve of breast cancer patients after treatment

[0028] Using online software (http: / / kmplot.com / analysis / ) analysis found that the survival curve of patients with high expression of SGCE in breast cancer patients who had undergone radiotherapy, chemotherapy and endocrine therapy corresponded to poor prognosis.

[0029] (2) Construction of shSGCE vector

[0030] 1) Design the shRNA sequence targeting SGCE, the sequence is as follows:

[0031]

[0032] 2) Synthesize positive and negative strand oligonucleotide sequences (oligos) through the company

[0033] 3) Dilute the synthesized forward and reverse oligos into 20 μM working solution for annealing

[0034] 4) The annealing system is as follows:

[0035]

[0036] Cook at 100°C for 4 minutes, then cool to room temperature naturall...

Embodiment 2

[0086] Example 2 Low expression of SGCE reduces enrichment of tumor stem cells caused by chemotherapy drugs

[0087] (1) Pretreatment of cells with chemotherapeutic drugs

[0088] 1) Prepare cells

[0089] Cells were counted after digestion and plated to 6-well plate, 3×10 per well 5 cells were cultured overnight at 37°C.

[0090] 2) Dosing treatment

[0091] The next day, the culture medium added with different chemotherapeutic drugs such as doxorubicin, paclitaxel and cisplatin was replaced, and the drug concentrations used were as follows:

[0092] Doxorubicin treatment of HCC1806 and MDA-MB-231 drug concentrations were 5nM and 1nM;

[0093] The drug concentrations of Paclitaxel treated HCC1806 and MDA-MB-231 were 1nM and 1nM respectively;

[0094] The drug concentrations of HCC1806 and MDA-MB-231 treated with cisplatin were 100nM and 100nM respectively;

[0095] In addition, a group of cells without any drug is required as a control.

[0096] The following experimen...

Embodiment 3

[0106] Example 3 SGCE knockdown promotes lysosomal degradation of EGFR

[0107] (1) Western detection

[0108] 1) Collection of protein samples

[0109] Cells grow to about 70%, remove the original medium, add basal medium to starve overnight, and stimulate with 50ng / ml EGF for 15min, 15min, 30min respectively and scrape the cells at each time point, add protein inhibitor containing protein lysis solution (Beiyuntian, P10013), lyse on ice for 30min, centrifuge at 12000g at 4°C for 15min, and transfer the protein sample in the upper layer to a new 1.5ml EP tube.

[0110] 2) Determination of protein concentration

[0111] Protein concentration was measured using BCA protein concentration assay kit (Pierce, 23225). According to the instructions, dilute the BSA mother solution to protein standards with different concentration gradients, and prepare the A+B mixed solution according to the proportion, and the mixed solution is prepared immediately for use. Pipette 10 μl of the p...

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Abstract

The invention belongs to the technical field of biology, and relates to application of a reagent for inhibiting an SGCE gene and a tumor chemotherapy drug to preparation of a drug for treating tumor chemotherapy and application of a reagent for inhibiting the SGCE gene to preparation of a drug for treating EGFR high-expression tumor. It is found that enrichment of tumor stem cells caused by chemotherapeutic drugs can be weakened after the SGCE gene is inhibited; after the SGCE is inhibited, the combination of the Cbl and the EGFR is enhanced; egfr degradation, finally, the sensitivity of the EGFR inhibitor-resistant cell strain to the EGFR inhibitor is remarkably enhanced, and it is prompted that the drug resistance of tumor cells to the EGFR inhibitor can be overcome when the inhibitor for synthesizing the SGCE and the EGFR inhibitor are jointly applied to drugs for treating EGFR high-expression tumors. Therefore, proliferation and metastatic growth of various EGFR high-expression tumors such as triple-negative breast cancer, non-small cell lung cancer, colorectal cancer and the like can be effectively inhibited by jointly inhibiting EGFR while inhibiting SGCE, and recurrence of cancer is effectively prevented. The reagent has important guiding significance in clinical application and has a wide prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of a reagent for inhibiting SGCE gene, in particular to the application of shRNA, siRNA and SGCE monoclonal antibody for inhibiting SGCE gene in the preparation of drugs for treating EGFR highly expressed tumors. Background technique [0002] There is a special class of cells in tumors—tumor stem cells. Cancer stem cells play an important role in tumor recurrence and metastasis. Chemotherapy drugs are a common way to treat tumors, but patients will develop drug resistance as the drug time increases. Chemotherapy drugs can kill dividing tumor cells, but have no obvious effect on quiescent tumor stem cells, and lead to the enrichment of tumor stem cells, which is one of the main reasons for drug resistance in patients. Screening molecules that can inhibit the enrichment of tumor stem cells during chemotherapy plays an important role in solving chemotherapy resistance. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/06A61K31/337A61K31/704A61K33/243A61K31/5377A61P35/00
CPCA61K31/337A61K31/5377A61K31/704A61K45/06A61P35/00A61K33/243
Inventor 焦保卫赵丽娜陈策实邱婷
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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