dCas9-sgLINC00261 system for inhibiting pancreatic cancer progression, construction method and application

A technology of dcas9-sglinc00261 and construction method, which is applied in the field of tumor molecular biology to enrich the regulatory mechanism and enhance the effect of inhibition

Inactive Publication Date: 2020-12-04
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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Problems solved by technology

Since methylation-mediated lncRNAs have not been reported on the function of PDAC, the use of targeted demethylation technology to activate the tumor suppressor potential of lnc

Method used

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  • dCas9-sgLINC00261 system for inhibiting pancreatic cancer progression, construction method and application
  • dCas9-sgLINC00261 system for inhibiting pancreatic cancer progression, construction method and application
  • dCas9-sgLINC00261 system for inhibiting pancreatic cancer progression, construction method and application

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Experimental program
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Embodiment

[0027] The construction method of dCas9-sgLINC00261 system comprises the following steps:

[0028] S1: Construct a stable CRISPR-dCas9 pancreatic cancer cell line using lentivirus; the sequence of the actual components of the lentivirus is EF1a-Dcas9-Tet1CD-CMV-EGFP, and the relevant steps are as follows:

[0029] A. Cell Plating

[0030] 90% confluent pancreatic cancer cells (CFPAC-1 and PANC-1 cell lines) were digested with trypsin to make a cell suspension, and the cell suspension (the number of cells was about 5×10 4 / mL) inoculated in 6-well, at 37°C, 5% CO 2 cultivated in an incubator;

[0031] B. Lentiviral Infection of Target Cells

[0032] After the cells in step A were cultured for 24 hours, when the degree of cell fusion reached about 30%, according to the MOI value of the cells (i.e. the multiplicity of infection value, the MOI values ​​of different cell lines of pancreatic cancer were different, the MOI value of PANC-1 was 10 , BXPC-3 and CFPAC-1 MOI values ​​...

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Abstract

The invention relates to the technical field of tumor molecular biology, and discloses a construction method of a dCas9-sgLINC00261 system. The method comprises the following steps: constructing a CRISPR-dCas9 pancreatic cancer cell line by using lentivirus, performing screening to obtain differential DNA methylation sites of a promoter region of LINC00261, constructing an sgRNA lentivirus vector,and transfecting the constructed sgRNA lentivirus vector into the CRISPR-dCas9 pancreatic cancer cell line. A dCas9-sgLINC00261 system is obtained. The invention also provides an application of the dCas9-sgLINC00261 system in preparation of drugs, markers or kits for preventing, detecting or treating pancreatic cancer. The cancer suppressor gene LINC00261 is modified through targeted demethylation, so that the endogenous expression quantity is increased, the pancreatic cancer proliferation and metastasis inhibiting capacity of the cancer suppressor gene is improved, and an innovative thoughtcan be provided for pancreatic cancer novel targeted drug research and development and clinical diagnosis and treatment.

Description

technical field [0001] The invention relates to the technical field of tumor molecular biology, in particular to a dCas9-sgLINC00261 system, construction method and application for inhibiting the progression of pancreatic cancer. Background technique [0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor of the digestive system. Due to its insidious onset and rapid progression, most patients have local invasion and distant metastasis at the first diagnosis, which is a high-risk disease in pancreatic cancer. major factor in mortality. Some studies predict that by 2030, pancreatic cancer will rise to the second leading cause of death from malignant tumors. Therefore, exploring the molecular mechanism of pancreatic cancer invasion and metastasis has important theoretical guiding significance and clinical application value for improving the diagnosis and treatment of pancreatic cancer. [0003] CRISPR / Cas9 is a technology that enables precise editing of s...

Claims

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Application Information

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IPC IPC(8): C12N15/867A61K48/00A61K38/46A61P35/00A61P1/18
CPCC12N15/86A61K48/0008A61K48/0058A61K38/465A61P35/00A61P1/18C12N2740/15043C12N2800/107C12N2830/008
Inventor 刘淞淞王槐志尹梁宇
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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