Osteosarcoma diagnosis and treatment preparation and application thereof

A technology of osteosarcoma and reagents, applied in the field of molecular biology, can solve the problem of stagnant 5-year survival rate of osteosarcoma

Inactive Publication Date: 2016-05-04
深圳贝安基因生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although clinical orthopedic workers have been committed to the research on its prevention and treatment, the overall 5-year survival rate of osteosarcoma has remained stagnant in recent years. Therefore, it is a long way to go to find effective targets for early and accurate diagnosis and treatment of osteosarcoma

Method used

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  • Osteosarcoma diagnosis and treatment preparation and application thereof
  • Osteosarcoma diagnosis and treatment preparation and application thereof
  • Osteosarcoma diagnosis and treatment preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The collection of embodiment 1 sample

[0057] 7 patients with primary osteosarcoma and 8 healthy controls. The patients with primary osteosarcoma and the healthy control group were required to fast for at least 12 hours. At 7:00-8:00 the next morning at room temperature, 10ml of venous blood was drawn into ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and peripheral blood was extracted. For mononuclear cell PBMCs, add 1ml Trizol reagent (Invitrogen), mix well, and store the specimen at -80°C for RNA extraction. All blood samples and pathological results should be authentic and reliable, the study was approved by the ethics committee, and the patients gave informed consent.

Embodiment 2

[0058] Example 2 total RNA extraction

[0059] Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of patients and healthy controls.

[0060] Requirements: RNA purity: OD260 / 280≧1.8, 28S / 18S≧1; RNA integrity: RIN value≧7.0.

[0061] Method: RNA concentration and purity detection method: NanoDrop2000;

[0062] RNA integrity detection method: Agilent2100 (RNA6000Nanokit), agarose gel electrophoresis (agarose gel concentration: 1% agarose gel; voltage: 5V / cm; time: 20min).

[0063] Small RNA sequencing sample requirements: concentration ≥ 200ng / μL, total amount ≥ 10μg of RNA (5 μg for a single library construction); OD260 / 280 between 1.8 and 2.2, OD260 / 230 ≥ 2.0, RIN ≥ 6.5, 28S :18S≥1.0, no RNA degradation.

Embodiment 3

[0064] Example 3 Sequencing and Data Analysis

[0065] The sequencing company carried out the establishment of the sequencing library and on-machine sequencing, and the sequencer used was the HiSeq2000 sequencer of Illumina Company.

[0066] The miRNA raw data were background-corrected by transcriptome data analysis software, and then the t-test was performed to obtain the P value, and then the Fisher test was used to combine the P values ​​to screen for differentially expressed miRNAs. Set p value2, and then artificially select and filter differentially expressed miRNAs up-regulated miR-6779-5p (log2(Fold_change) value is 2.16) into our research scope.

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Abstract

The invention relates to an osteosarcoma diagnosis and treatment preparation and application thereof, in particular to new application of mir-6779 and/or mature miRNA of mir-6779 in diagnosing and treating osteosarcoma. By carrying out sequencing analysis, miRNA expression data is obtained, then bioinformatics analysis is carried out, and it is found that high expression of mir-6779 in an osteosarcoma group is obvious; it is shown through further molecular biological verification results that miR-6779-5p is closely related to osteosarcoma, the multiplication capacity of osteosarcoma cells can be effectively reduced by inhibiting miR-6779-5p expression, and the osteosarcoma diagnosis and treatment preparation can be used for clinical diagnosis, prevention and treatment, and has good practical application value.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a preparation for the diagnosis and treatment of osteosarcoma and its application, and more specifically to the new application of mir-6779 and / or its mature miRNA in the diagnosis and treatment of osteosarcoma. Background technique [0002] Osteosarcoma (Osteeosarcoma) is the most common malignant primary tumor of bone except plasmacytoma, and its histological feature is that in most cases the tumor cells produce osteoid or immature bone. The disease tends to occur in the long bone period of adolescents, without obvious clinical symptoms at the onset, and is easily confused with trauma, and has a high degree of malignancy, so it is very harmful and has a high mortality rate. Although clinical orthopedic workers have been committed to the research on its prevention and treatment, the overall 5-year survival rate of osteosarcoma has remained stagnant in recent years. There...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088A61P35/00C12Q1/68
CPCA61K31/7088A61K48/0066C12Q1/6886C12Q2600/178
Inventor 何春耒高辉刘午阳黄为民刘胜吴东保何澄徐房添杨亚东
Owner 深圳贝安基因生物科技有限公司
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