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Application of GPR115 gene in preparation of anti-lung cancer drug and diagnostic kit thereof

A kit, gpr115-shrna-3 technology, applied in disease diagnosis, gene therapy, biological testing, etc., can solve the problems of lack of universality and effective experimental demonstration, and achieve the effect of reducing invasion ability and migration

Active Publication Date: 2020-01-17
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this is only a prediction conclusion of a database, lacking universality and effective experimental evidence to illustrate

Method used

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  • Application of GPR115 gene in preparation of anti-lung cancer drug and diagnostic kit thereof
  • Application of GPR115 gene in preparation of anti-lung cancer drug and diagnostic kit thereof
  • Application of GPR115 gene in preparation of anti-lung cancer drug and diagnostic kit thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Through the data analysis of TCGA and Oncomine database, the expression level map of GPR115 mRNA in lung cancer tissues is obtained, as shown in figure 1 As shown, it indicated that GPR115 mRNA was increased in lung adenocarcinoma and lung squamous cell carcinoma, and the increase was more significant in stage IV tumors.

[0028]Tissue samples: 299 patients with lung cancer who underwent surgical treatment in the Affiliated Hospital of Nantong University from January 2005 to April 2011 were included, and their medical records and lung cancer tissue samples (including 156 cases of adenocarcinoma, 95 cases of squamous cell carcinoma, and adenosquamous cell carcinoma) were collected. 47 cases). Among the 299 patients, there were 198 males and 101 females, ranging in age from 35 to 83 years old. The pathological staging of lung cancer is the eighth edition of the TNM staging standard for lung cancer released by the Union for International Cancer Control (UICC) in 2017. In...

Embodiment 2

[0047] siRNA design:

[0048] For the GPR115 gene, China Nanjing Biomics Company was commissioned to design three small interfering RNA sequences, which are:

[0049] hs-GRP115-si-1: 5′-GAUCCAAGAUUCACCUAAAdTdT-3′:

[0050] hs-GRP115-si-2: 5'-GGAUUUACAUGUAAUCAAAdTdT-3';

[0051] hs-GRP115-si-3: 5'-CAUUGAGAGUGUAGCUCAAdTdT-3';

[0052] siRNA-NC: 5'-UUCUCCGAACGUGUCACGUdTdT-3.

[0053] Culture of lung cancer cell lines:

[0054] Four lung cancer cell lines H1650, A549, H1975 and SPCA were cultured in 10% RPMI 1640 medium, and the environment was maintained at 37°C, 5% CO 2 . Change the medium every 1-2 days and select cells in the logarithmic growth phase for experiments.

[0055] siRNA transfection:

[0056] (1) Five control groups were set up, namely: normal cell group (CK), silencing NC group (siRNANC), silencing siRNA 1 group (hs-GPR115-si-1), silencing siRNA2 group (hs-GPR115-si- 2) and silencing siGPR115 3 groups (hs-GPR115-si-3);

[0057] (2) According to the amount...

Embodiment 3

[0089] CCK8 assay to detect cell proliferation:

[0090] (1) H1650 and SPCA cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 3×10 4 cells / mL, seeded in 96-well culture plate, 100 μL per well, 37°C, 5% CO 2 Cultivate in the incubator for 24 hours;

[0091] (2) Three experimental groups: CK group, siRNANC group and hs-GRP115-si-2 group;

[0092] (3) Treat the cells in groups according to the experiment, and after culturing for 24h, 48h and 72h respectively, add 10 μL of CCK8 to each well, at 37°C, 5% CO 2 Cultivate in the incubator and incubate for 4 hours in the dark;

[0093] (4) Measure the OD value at the same time point at the 492nm wavelength of the microplate reader, and use the measured OD value to analyze the changes in cell growth;

[0094] (5) GraphPad Prism 6 was used to draw the cell growth curve.

[0095] Results: Compared with the CK group, GPR115 silencing could inhibit cell proliferation in a time-dependent manner...

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Abstract

The invention discloses application of a GPR115 gene in preparation of an anti-lung cancer drug and a diagnostic kit thereof, and belongs to the technical field of cancer precision medical drugs. Immunohistochemistry proves that the expression of GPR115 in lung cancer is increased through a lung cancer clinical sample tissue chip, and GPR115 is related to prognosis; the influence of down-regulation of GPR115 gene expression by siRNA on biological behaviors such as proliferation and invasion of lung cancer cells is studied through an in-vitro cell experiment, it is found that the specific siRNAsequence can effectively inhibit the expression of GPR115 protein in human lung cancer cell strains H1650 and SPCA, and after the expression of GPR115 is inhibited by siRNA, the proliferation of H1650 and SPCA cells is slowed down, and the cell invasion ability is reduced. The GPR115, as a target for lung cancer gene therapy, can be widely applied to preparation of a diagnostic kit for precisionmedical treatment and treatment of lung cancer with high expression of GPR115.

Description

technical field [0001] The invention belongs to the technical field of precision medicine for cancer, and more specifically relates to the application of GPR115 gene in the preparation of anti-lung cancer medicine and its diagnostic kit. Background technique [0002] Lung cancer (Lung cancer) is a malignant tumor with strong invasiveness and rapid progress due to the uncontrolled growth of lung tissue cells. According to the latest global cancer statistics, lung cancer is still the main cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) is the most common, accounting for 80%-85%. It is often incurable because most patients are not diagnosed until late, out of therapeutic value, or inoperable. The treatment of lung cancer is closely related to the cell type, the extent of spread and the performance status of the person. Common ones include: palliative care, surgery, chemotherapy and radiotherapy. The emergence of targeted therapy and immunotherapy in re...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/574C12N15/113A61K31/7105A61K48/00A61P35/00
CPCG01N33/68G01N33/57423C12N15/1135A61K31/7105A61P35/00C12N2310/14G01N2800/52G01N2333/726
Inventor 黄剑飞张筱静王营静
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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