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Application of protein deacetylase SIRT1 in regulating radiosensitivity of mouse prostate cancer RM-1 cells

A technology of deacetylase and prostate cancer, which is applied in the field of medicine and can solve the controversy about the regulation of radiosensitivity of prostate cancer.

Pending Publication Date: 2022-05-13
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The expression of SIRT1 is increased in prostate cancer, but its role in the development of prostate cancer and the regulation of radiosensitivity is still controversial

Method used

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  • Application of protein deacetylase SIRT1 in regulating radiosensitivity of mouse prostate cancer RM-1 cells
  • Application of protein deacetylase SIRT1 in regulating radiosensitivity of mouse prostate cancer RM-1 cells
  • Application of protein deacetylase SIRT1 in regulating radiosensitivity of mouse prostate cancer RM-1 cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. SIRT1 overexpression

[0029] S1. Inoculate mouse prostate cancer RM-1 cells in a six-well plate at a density of 1.5×10 5 / well, placed at 37°C, 5% CO 2 Grow in an incubator for 24 hours, and the medium used is RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin;

[0030] S2. Mix 2.5 μg of SIRT1 plasmid and negative control with liposome transfection reagent respectively, and let stand for 10 minutes;

[0031] S3. Replace the medium with opti-MEM medium, add the plasmid solution dropwise to the culture dish, and incubate for 6 hours;

[0032] S4. Replace with RPMI-1640 complete medium without antibiotics, and irradiate the cells with X-RAD 225 irradiator at a dose of 16Gy and a dose rate of 2Gy / min. After the irradiation, the cells continue to grow for 24 hours.

Embodiment 2

[0034] 1. SIRT1 knockdown

[0035] S1. Spread mouse prostate cancer RM-1 cells on a six-well plate at a density of 1.5×10 5 / well, placed at 37°C, 5% CO 2 Grow in an incubator for 24 hours, and the medium used is RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin;

[0036] S2. Mix 100 nM SIRT1 small interfering RNA and negative control with liposome transfection reagent respectively, and let stand for 10 minutes. SIRT1 small interfering RNA sense strand: 5’-GCACCGAUCCUCGAACAAUTT-3’; SIRT1 small interfering RNA antisense strand: 5’-AUUGUUCGAGGAUCGGUGCTT-3’;

[0037] S3. Replace the medium with opti-MEM medium, add the plasmid solution dropwise to the culture dish, and incubate for 6 hours;

[0038] S4. Replace with RPMI-1640 complete medium without antibiotics, and irradiate the cells with X-RAD 225 irradiator at a dose of 16Gy and a dose rate of 2Gy / min. After the irradiation, the cells continue to grow for 24 hours.

Embodiment 3

[0040] 1. Detection of protein expression by Western blot

[0041] 1.1 Lyse RM-1 cells with RIPA lysis buffer containing 1% protease and phosphatase inhibitors;

[0042] 1.2 Scrape the cells and collect them into a 1.5ml centrifuge tube, shake for 15 seconds, and ice-bath for 10 minutes, repeat 3 times;

[0043] 1.3 Centrifuge: 12000g, 30 minutes, 4°C, absorb the supernatant.

[0044] 1.4 The total protein concentration is determined by BCA detection kit;

[0045] 1.5 After the total protein concentration is quantified and balanced, mix it with protein loading buffer at a ratio of 4:1, and denature at 100°C for 5 minutes;

[0046] 1.6 The protein is separated by 7.5% or 12% SDS-PAGE gel;

[0047] 1.7 Transfer to 0.22μm PVDF membrane by semi-dry transfer instrument;

[0048] 1.8 Block the membrane with 5% bovine serum albumin at room temperature for 1 hour, wash the membrane with TBST for 10 minutes, 3 times;

[0049] 1.9 Incubate the membrane with SIRT1 and cleaved-caspase3...

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Abstract

The invention discloses an application of protein deacetylase SIRT1 in regulating the radiosensitivity of a mouse prostate cancer RM-1 cell, and particularly relates to an application of overexpression or knock-down protein deacetylase SIRT1 in regulating the radiosensitivity of the mouse prostate cancer RM-1 cell. The radiosensitivity of the RM-1 cells can be weakened by increasing the expression of the SIRT1, and the radiosensitivity of the RM-1 cells can be enhanced by reducing the expression of the SIRT1; the method specifically comprises the steps that after RM-1 cells are subjected to overexpression or SIRT1 knockout and are subjected to 16Gy X-ray irradiation, western blot verifies the expression of apoptosis-related protein cleaved-caspase3, and the result shows that the protein expression of the cleaved-caspase3 is increased due to irradiation, the protein expression of the cleaved-caspase3 is reduced due to overexpression of the SIRT1, and the protein expression of the cleaved-caspase3 is increased due to knockout of the SIRT1; after RM-1 cells overexpress or knock down SIRT1 and receive 16Gy X-ray irradiation, apoptotic cells are detected through flow cytometry, and the result shows that the apoptotic cells are increased due to irradiation, the apoptotic cells are reduced due to overexpression of SIRT1, and the apoptotic cells are increased due to knock down of SIRT1.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to the application of protein deacetylase SIRT1 in regulating the radiosensitivity of mouse prostate cancer RM-1 cells. Background technique [0002] The protein deacetylase SIRT1 is a member of the Sirtuin protein family and is a highly conserved nicotinamide adenine dinucleotide (NAD + )-dependent sirtuin. SIRT1 can consume NAD in enzymatic reactions + And generate NAM, which removes acetyl groups from many protein substrates, thereby altering transcriptional activity. SIRT1 can participate in the regulation of various physiological processes, such as metabolism, aging, apoptosis, autophagy, inflammation, and DNA repair; SIRT1 is also involved in pathological processes such as heart disease, diabetes, and cancer. [0003] The expression of SIRT1 is different in different tumor types, and its dysregulation has a certain impact on the occurrence and development of tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N5/10C12N15/55C12N15/113C12N15/85
CPCC12N9/80C12N15/1137C12N15/85C12N5/0693C12N5/0683C12Y305/01098C12N2510/00C12N2310/14
Inventor 罗兰王凯旋晏琛齐素华蓝婷陶艾彬黄琳燕王婉崔雯雯杨旭
Owner XUZHOU MEDICAL UNIV