A stable cell line of du145 with low expression of gfpt1 and its construction and application

A DU145, low-expression technology, applied in the field of cell biology, biochemistry and molecular biology, can solve the problems of the survival period and overall survival of triple-negative breast cancer patients.

Active Publication Date: 2022-08-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

At the same time, studies have confirmed that the survival period and overall survival of patients with triple-negative breast cancer with high expression of GFPT1 are significantly reduced

Method used

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  • A stable cell line of du145 with low expression of gfpt1 and its construction and application
  • A stable cell line of du145 with low expression of gfpt1 and its construction and application

Examples

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preparation example Construction

[0032] Preparation of protein samples

[0033] 1) Prepare RIPA protein lysis solution (100ul per 600mm petri dish) and add protease inhibitors.

[0034] 2) Collect the cells, wash the cells with pre-cooled PBS, blow off the cells with the prepared protein lysis solution, transfer the cells to a centrifuge tube, put them into a DNA mixer and mix well, 4°C for 30 min.

[0035] 3) Put the centrifuge tube into a centrifuge, 13,000 rpm at 4°C for 15 minutes, and transfer the supernatant to another centrifuge tube.

[0036] 4) Determination of protein concentration by BCA method

[0037] Blank: RIPA

[0038] Mix 20μl of BSA with different concentrations (0.125μg / μl, 0.25μg / μl, 0.5μg / μl, 1μg / μl, 2μg / μl) to prepare a standard and dilute 2μl of the sample to 18μl of RIPA.

[0039] BCA system: A:B=50:1 ratio to configure the measurement solution, finally according to 200μl BCA + 20μl sample or standard, after shaking, incubate at 37°C for 30min, and measure the protein concentration ...

example 1

[0072] Example 1 GFPT1 promotes tumor cell proliferation.

[0073] The proliferation efficiency of tumor cells was detected by crystal violet staining in DU145 cells differentially expressing GFPT1. 3000 cells were seeded in a twelve-well plate, and the cells were taken out on the 3rd, 5th, 7th, and 9th day for crystal violet staining. The cells were washed with acetic acid, and the values ​​were read with a microplate reader to draw the cell proliferation curve. like figure 1 Low expression of GFPT1 is shown to inhibit cell proliferation.

example 2

[0074] Example 2 GFPT1 regulates AMPK phosphorylation.

[0075] The constructed cell line with differential expression of GFPT1 was used to verify the effect of GFPT1 on AMPK phosphorylation. The cells of differential expression of GFPT1 and the corresponding control group were subjected to AMPK activation (such as adding 2-DG treatment), and AMPK antibody was used by WesternBlot. And AMPK Thr172 phosphorylation antibody, and AMPK-regulated ACC signaling pathway antibody, observe the change trend of AMPK protein, AMPK phosphorylation and corresponding signaling pathway in cells, and prove that GFPT1 regulates AMPK signaling pathway such as figure 2 shown.

[0076] The above experiments show that GFPT1 has an important biological function in the occurrence and development of tumors and can regulate the phosphorylation of AMPK. Therefore, low expression of GFPT1 can inhibit the growth of prostate cancer, which is helpful for GFPT1 in cancer diagnosis, prognosis, treatment, and ...

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Abstract

The invention discloses a newly discovered pathway-GFPT1 regulating AMPK phosphorylation pathway that can affect the proliferation of prostate cancer. Specifically, shRNA interference technology was used to interfere with GFPT1 in DU145 cells to activate the activity of AMPK and inhibit cell proliferation. Crystal violet staining and Western blotting experiments confirmed that the inhibition of GFPT1 can activate the AMPK pathway and inhibit cell proliferation, thereby achieving The purpose of effective treatment of prostate cancer.

Description

technical field [0001] The invention relates to cell biology, biochemistry and molecular biology technology, in particular to a method for detecting cell proliferation and AMPK phosphorylation. Background technique [0002] The occurrence and development of tumors are often associated with the imbalance of a series of energy-generating mechanisms. During the evolution from normal cells to tumor cells, cells have to face various stresses, such as activation of oncogenes, hypoxic conditions, lack of nutrients, and chemotherapy and radiation therapy. Under stress, tumor cells provide nutrients that support tumor survival and growth by regulating cellular metabolism. One of the most representative molecules linking cellular metabolism and tumor is the AMP-activated protein kinase AMPK (AMP-activated protein kinase, AMPK). [0003] Disturbance of tumor metabolism is an important feature of tumorigenesis and development. Tumor growth is often associated with an imbalance of a se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 朴海龙刘静
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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