YAP1 gene and application of inhibitor in enhancing chemosensitivity of stem cell of bladder cancer

Abstract

The invention discloses a YAP1 gene and application of an inhibitor in enhancing chemosensitivity of a stem cell of bladder cancer. The YAP1 gene is related to chemosensitivity of the stem cell of bladder cancer. The inhibitor where the YAP1 gene is highly expressed can improve the chemosensitivity of the stem cell of bladder cancer on chemotherapeutics mitomycin C and cis-platinum by down-regulating the expression of the YAP1 gene, so that the inhibiting rate of the chemotherapeutics mitomycin C and cis-platinum on the stem cell of bladder cancer is increased. The inhibitor where the YAP1 gene is highly expressed can be any one or more of siRNA, shRNA, dsRNA, miRNA, cDNA, a small molecular compound, a peptide and an antibody, wherein the small molecular compound is a 2-amido benzoxazole derivative.

Description

technical field

[0001] The invention belongs to the field of biochemistry, and relates to the discovery of targets related to the chemosensitivity of bladder cancer stem cells and the development of target inhibitors, in particular to the application of YAP1 gene and inhibitors in enhancing the chemosensitivity of bladder cancer stem cells. Background technique

[0002] Cancer stem cells refer to cells in tumors that have unlimited self-renewal ability and can generate heterogeneous tumor cells. Studies have found that such cells are closely related to tumor growth, metastasis and recurrence. Many properties of these cells are similar to stem cells, so they are called tumor stem cells. The theory of cancer stem cells believes that the root cause of tumors is the formation of tumor stem cells in body tissues, and the recurrence and drug resistance after tumor treatment are also related to tumor stem cells. Compared with ordinary tumor cells, tumor stem cells have a high abi...

Examples

Embodiment 1

[0025] Example 1: Sorting of Bladder Cancer Stem Cells

[0026] 1. Experimental materials

[0027] Human bladder cancer T24 cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin), incubator temperature 37°C, CO2 concentration 5%, every 2 Change the solution once a day.

[0028] The cell inhibition rate experiment used CCK-8 cell proliferation and cytotoxicity detection kit: according to the instructions, the more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color, the depth of the color and the number of cells linear relationship.

[0029] Cell cycle detection was detected using a cell cycle point-of-care detection kit, followed by flow cytometry analysis.

[0030] 2. Experimental method

[0031] 1. Chemotherapy drug intervention culture sorting drug-resistant bladder cancer cells

[0032] Inoculate 5×10 T24 cells in T75 culture flask...

Embodiment 2

[0047] Example 2: Expression of YAP1 gene in bladder cancer stem cells and its relationship with chemotherapy sensitivity

[0048] 1. Experimental materials

[0049] Human bladder cancer T24 cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin), incubator temperature 37°C, CO2 concentration 5%, every 2 Change the solution once a day.

[0050] Bladder cancer stem cells were sorted by the method in Example 1 to obtain drug-resistant cells.

[0051] The siRNA targeting YAP1 and the siRNA negative control were provided by Shanghai Gemma.

[0052] 2. Experimental method

[0053] 1. Cell culture and transfection

[0054] Drug-resistant cells were collected and inoculated on culture plates, and randomly divided into blank control group, siRNA interference group and siRNA negative control group. The blank control group was not transfected; the siRNA interference group was transfected with siRNA tha...

Embodiment 3

[0066] Example 3: Effects of Small Molecular Compounds on Chemosensitivity of Bladder Cancer Stem Cells

[0067] 1. Experimental materials

[0068] Relevant experimental material is the same as embodiment 1 and embodiment 2.

[0069] 2. Experimental method

[0070] 1. Cell culture and grouping

[0071] Drug-resistant cells were collected, and after being cultured with RPMI-1640 medium containing 5% fetal bovine serum for 4 hours, they were randomly divided into blank control group and experimental group 1-3. The sensitizer continued to intervene and cultivate for 12 hours.

[0072] The chemical structure and concentration in the culture medium of each group of sensitizers in table 1

[0073]

[0074] 2. RT-PCR determination of YAP1 expression in bladder cancer stem cells

[0075] After 12 hours of intervening culture, collect the drug-resistant cells of the blank control group and the experimental group 1-3, wash with PBS, centrifuge and discard the supernatant, use th...

technical field

[0001] The invention belongs to the field of biochemistry, and relates to the discovery of targets related to the chemosensitivity of bladder cancer stem cells and the development of target inhibitors, in particular to the application of YAP1 gene and inhibitors in enhancing the chemosensitivity of bladder cancer stem cells. Background technique

[0002] Cancer stem cells refer to cells in tumors that have unlimited self-renewal ability and can generate heterogeneous tumor cells. Studies have found that such cells are closely related to tumor growth, metastasis and recurrence. Many properties of these cells are similar to stem cells, so they are called tumor stem cells. The theory of cancer stem cells believes that the root cause of tumors is the formation of tumor stem cells in body tissues, and the recurrence and drug resistance after tumor treatment are also related to tumor stem cells. Compared with ordinary tumor cells, tumor stem cells have a high abi...

Examples

Embodiment 1

[0025] Example 1: Sorting of Bladder Cancer Stem Cells

[0026] 1. Experimental materials

[0027] Human bladder cancer T24 cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin), incubator temperature 37°C, CO2 concentration 5%, every 2 Change the solution once a day.

[0028] The cell inhibition rate experiment used CCK-8 cell proliferation and cytotoxicity detection kit: according to the instructions, the more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color, the depth of the color and the number of cells linear relationship.

[0029] Cell cycle detection was detected using a cell cycle point-of-care detection kit, followed by flow cytometry analysis.

[0030] 2. Experimental method

[0031] 1. Chemotherapy drug intervention culture sorting drug-resistant bladder cancer cells

[0032] Inoculate 5×10 T24 cells in T75 culture flask...

Embodiment 2

[0047] Example 2: Expression of YAP1 gene in bladder cancer stem cells and its relationship with chemotherapy sensitivity

[0048] 1. Experimental materials

[0049] Human bladder cancer T24 cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin), incubator temperature 37°C, CO2 concentration 5%, every 2 Change the solution once a day.

[0050] Bladder cancer stem cells were sorted by the method in Example 1 to obtain drug-resistant cells.

[0051] The siRNA targeting YAP1 and the siRNA negative control were provided by Shanghai Gemma.

[0052] 2. Experimental method

[0053] 1. Cell culture and transfection

[0054] Drug-resistant cells were collected and inoculated on culture plates, and randomly divided into blank control group, siRNA interference group and siRNA negative control group. The blank control group was not transfected; the siRNA interference group was transfected with siRNA tha...

Embodiment 3

[0066] Example 3: Effects of Small Molecular Compounds on Chemosensitivity of Bladder Cancer Stem Cells

[0067] 1. Experimental materials

[0068] Relevant experimental material is the same as embodiment 1 and embodiment 2.

[0069] 2. Experimental method

[0070] 1. Cell culture and grouping

[0071] Drug-resistant cells were collected, and after being cultured with RPMI-1640 medium containing 5% fetal bovine serum for 4 hours, they were randomly divided into blank control group and experimental group 1-3. The sensitizer continued to intervene and cultivate for 12 hours.

[0072] The chemical structure and concentration in the culture medium of each group of sensitizers in table 1

[0073]

[0074] 2. RT-PCR determination of YAP1 expression in bladder cancer stem cells

[0075] After 12 hours of intervening culture, collect the drug-resistant cells of the blank control group and the experimental group 1-3, wash with PBS, centrifuge and discard the supernatant, use th...

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