Ku70 protein T455 site phosphorylation inhibitor and application thereof

A phosphorylation inhibitor and site technology, applied in the field of Ku70 protein T455 site phosphorylation inhibitor, can solve the problems of high mortality, treatment failure of cancer patients, ineffective prolongation of survival time and overall survival rate of cancer patients, etc. achieve good specificity

Active Publication Date: 2020-12-15
中国人民解放军总医院第八医学中心
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the current clinical targeted cancer treatment methods are constantly innovating, surgical treatment combined with radiotherapy and chemotherapy is still the first choice for clinical treatment of lung cancer patients, but the high postoperative recurrence and metastasis rates are leading to treatment failure and high mortality in clinical cancer patients. The main reason for the high rate, it does not effectively prolong the survival time and overall survival rate of cancer patients

Method used

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  • Ku70 protein T455 site phosphorylation inhibitor and application thereof
  • Ku70 protein T455 site phosphorylation inhibitor and application thereof
  • Ku70 protein T455 site phosphorylation inhibitor and application thereof

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Experimental program
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Effect test

Embodiment 1

[0047]Taking non-small cell lung adenocarcinoma A549 cells and H1299 cells as the research objects, the effects of different expression levels of ING5 on Ku70 expression and Ku70 T455 phosphorylation levels were observed by quantitative modified phospho-omics, and Ku70 in each cell line was detected by Western blot. Expression changes at protein level and T455 phosphorylation level. As shown in Table 1, quantitative modified phospho-omics studies found that ING5 overexpression can significantly inhibit the phosphorylation of Ku70 threonine T455; Figure 1-2 As shown, the results of Western blot showed that overexpression of ING5 inhibited the phosphorylation of Ku70 T455, and the phosphorylation level of Ku70 T455 was increased in lung cancer cells with ING5 gene knockdown, which further verified the results of quantitative proteomics.

[0048] Table 1 Overexpression of ING5 down-regulates Ku70 threonine T455 phosphorylation

[0049]

Embodiment 2

[0051] Construction of Ku70 stable knockdown (KD), knockdown complement wild-type (WT), T455 phosphorylation deficient (T455A) and T455 phosphorylation mimic (T455D) cell lines on non-small cell lung adenocarcinoma A549-ING5-OE cells , Western blot detection of Ku70 protein level and T455 phosphorylation level in each cell line. Specifically as follows:

[0052] (1) Synthesize 3 siRNAs against the human ku70 gene, transfect the A549-ING5-puro stable transfection line, detect the expression of Ku70 mRNA in the transfected cells by RT-PCR, and select the sequence with the best interference efficiency. The shRNA lentiviral vector was constructed, and the A549-ING5-puro stable transfection was transfected with the lentivirus packaged in 293T cells, and the positive cells were screened with G418 antibiotics to obtain ku70 stable knockdown cells. According to the siRNA sequence with the best interference effect, 3-4 codons corresponding to the siRNA sequence of Ku70 T455A and T455D...

Embodiment 3

[0058] Observe the proliferation ability of each cell line constructed in the foregoing Example 2 by CCK8 cell proliferation experiment: the experiment is carried out in a 96-well plate, according to 1×10 per well 3 Cells were plated, and cell proliferation was detected at 24h, 48h, 72h, 96h and 120h after plating. When performing experiments, first discard the medium, add 100 μL of fresh medium to each well, and then add 10 μL of CCK8 solution, at 37°C, 5% CO 2 Incubate in an incubator for 4 h, and detect the absorbance at 450 nm with a microplate reader.

[0059] like Figure 5 As shown, overexpression of ING5 significantly inhibited the proliferation of Ku70 knockdown and complement wild-type (WT) cells and T455 phosphorylation mimic (T455D) cells, while the proliferation of Ku70 T455 phosphorylation-deficient (T455A) cells was significantly enhanced.

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Abstract

The invention relates to a Ku70 protein T455 site phosphorylation inhibitor and application thereof, in particular to application of the Ku70 protein T455 site phosphorylation inhibitor in preparationof a reagent for inhibiting cancer invasion and metastasis, a method for inhibiting intracellular Ku70 protein T455 site phosphorylation and cancer cells with inhibited invasion and metastasis capacity; and a new treatment way and a new research model are provided for cancer invasion and metastasis, especially lung cancer invasion and metastasis.

Description

technical field [0001] The invention relates to the field of cancer invasion and metastasis, in particular to an inhibitor of Ku70 protein T455 site phosphorylation and application thereof. Background technique [0002] Cancer is currently the number one killer of human beings, and its morbidity and mortality are increasing year by year, becoming a disease that seriously threatens human health and life. Invasion and metastasis is the main cause of high mortality in cancer patients, in which oncogene activation and tumor suppressor gene inactivation are important causes of cancer invasion and metastasis. Although the current clinical targeted cancer treatment methods are constantly innovating, surgical treatment combined with radiotherapy and chemotherapy is still the first choice for clinical treatment of lung cancer patients, but the high postoperative recurrence and metastasis rates are leading to treatment failure and high mortality in clinical cancer patients. The main ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12Q1/02C12N5/10A61K45/00A61P35/00
CPCG01N33/68G01N33/5005C12N5/0693C07K14/4703A61K45/00A61P35/00
Inventor 孟瑾李卓王玥赵冠人陈明车玲
Owner 中国人民解放军总医院第八医学中心
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