Application of human GRK5 genes

A gene and function technology, applied in the field of new use of human GRK5 gene, can solve the problem of unclear function of non-small cell lung cancer

Active Publication Date: 2018-07-06
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, until now, the function of GRK5 in NSCLC remains unclear

Method used

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  • Application of human GRK5 genes
  • Application of human GRK5 genes
  • Application of human GRK5 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Real-time fluorescent quantitative PCR experiment to detect the expression of GRK5 in non-small cell lung cancer cell lines

[0040] 1. Extraction of total cell RNA

[0041] (1) When normal lung bronchial epithelial cells and non-small cell lung cancer cells grow well, remove the supernatant, wash off the serum with PBS, add 1mL TRizol, and let stand for 5 minutes to ensure that Trizol fully lyses the cells, and pipette the cells from the culture dish When it comes down, the liquid is transferred to the centrifuge tube, pipetting repeatedly until there is no obvious large precipitation; let it stand at room temperature for 5 minutes;

[0042] (2) Turn on the 4°C centrifuge in advance, centrifuge at 12,000g for 5 minutes, and transfer the supernatant to a new 1.5mL centrifuge tube;

[0043] (3) Add 200μL of chloroform, vortex, centrifuge at 12,000g for 15min in a centrifuge at 4°C, after which the liquid is divided into three layers;

[0044] (4) Take the upper water p...

Embodiment 2

[0062] Example 2: Western blot detection of GRK5 protein expression

[0063] 1. WB (Western Blotting) detection

[0064] 1.1 Extraction of total cell protein

[0065] After processing the cells according to the specific experiment, discard the supernatant medium and wash with PBS once; add the corresponding cell lysate according to the amount of cell pellet, and repeat the freeze-thaw lysis twice; centrifuge at 4°C, 12000 rpm for 10 min, and take The supernatant was discarded and the pellet was used for subsequent experiments.

[0066] 1.2 Protein concentration detection and denaturation treatment

[0067] The protein concentration detection kit is the Biyuntian BCA protein concentration determination kit (enhanced), item number: P0010S. Methods as below:

[0068] Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the standard to the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μL so that the concentration of the standard is 0, 0.025, respectively , 0.05, 0.1, 0...

Embodiment 3

[0073] Example 3: Clinical experiment

[0074] 1. Immunohistochemistry experiment

[0075] Non-small cell lung cancer tissue immunohistochemistry was obtained in cooperation with Director Fan Songqing of the Department of Pathology, Xiangya Second Hospital of Changsha, Hunan. 638 clinical samples were placed in 80 ℃ baking sheets for 2 hours, soaked in xylene and dewaxed, dehydrated by gradient alcohol, and immersed Remove tissue peroxidase in 0.3% hydrogen peroxide (prepared in methanol) solution for 15 minutes, rinse with Tris buffer saline (TBS), microwave at 95°C for 20 minutes, restore antigen for 1 hour at room temperature; then wash three times with TBS, 5% Goat serum was incubated for 30 minutes to remove non-specific binding. The primary antibody anti-GRK5 (Santa Cruz Biotechnology, Cat# sc-565) was diluted to 1:200 with blocking solution, incubated at 4°C for two hours, washed with TBS three times, and HRP-labeled secondary antibody (Santa cruz Biotechnology, USA) Incuba...

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Abstract

The invention discloses application of human GRK5 genes, namely application of human GRK5 genes serving as cancer cell specified action targets in preparation of drugs for treating non-small cell lungcancer. The cancer cell specified action targets are RNA interference effect targets. Compared with normal pulmonary bronchial epithelial cells, the GRK5 genes have high expressions in the non-smallcell lung cancer cells; RNA interfering lentivirus designed aiming at the GRK5 genes can inhibit GRK5 expression and is capable of obviously inhibiting proliferation of the non-small cell lung cancercells, blocking tumor cell division at a G2/M phase, promoting cells apoptosis, inhibiting cell migration and inhibiting formation of xenograft tumor in nude mice, and the aim of treating the non-small cell lung cancer can be further achieved. The human GRK5 genes can serve as treatment targets of the non-small cell lung cancer, and provide wide prospects for developing medicines in treatment of the non-small cell lung cancer based on the GRK5 genes in future.

Description

Technical field [0001] The invention relates to a new use of a gene, especially a new use of the human GRK5 gene. Background technique [0002] Non-small cell lung cancer is one of the world's leading malignant tumors that cause death due to cancer, and non-small cell lung cancer is the most among all lung cancer cases, accounting for about 80%. Non-small cell lung cancer mainly includes three types: squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Although many treatments have been developed, the average survival time of patients with non-small cell lung cancer is less than one year. According to reports, the epidermal growth factor receptor-tyrosine kinase combined inhibitor is more effective for some patients with L858 or exon 19 deletion EGFR mutation. However, the epidermal growth factor receptor-tyrosine kinase combined inhibitors are becoming more and more serious in congenital and acquired tolerance, so there is an urgent need to develop a new generatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00A61P35/04
CPCA61K48/005
Inventor 陈勇彬杨翠萍江丽萍熊秋霞
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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