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Blood circulation exosome RNA based detection for gene mutation related to osteosarcoma pulmonary metastasis and application thereof

A technology of osteosarcoma and exosomes, applied in the field of clinical medicine, can solve the problems of less than 20% survival rate, difficulty in obtaining biopsy materials, heterogeneity of lung metastases, and poor prognosis

Inactive Publication Date: 2018-11-27
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

[0002] The survival rate of osteosarcoma with lung metastasis is less than 20%, and the prognosis is extremely poor
However, due to the difficulty in early detection of osteosarcoma lung metastasis, the difficulty in obtaining biopsy materials, and the heterogeneity of lung metastases, there is currently no effective method for the detection of molecular targets in metastatic osteosarcoma.

Method used

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  • Blood circulation exosome RNA based detection for gene mutation related to osteosarcoma pulmonary metastasis and application thereof
  • Blood circulation exosome RNA based detection for gene mutation related to osteosarcoma pulmonary metastasis and application thereof
  • Blood circulation exosome RNA based detection for gene mutation related to osteosarcoma pulmonary metastasis and application thereof

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Embodiment 1

[0022] Methods: The supernatants of two pairs of osteosarcoma cell lines, MG63.2 (with lung metastasis), MG63 (without lung metastasis), HOS / 143B (with lung metastasis), HOS / MNNG (without lung metastasis), and two osteosarcoma cell lines were collected. The serum of patients before lung metastasis (only tumor in situ) and after metastasis (only metastatic tumor). Extracellular vesicles were isolated and purified by ultracentrifugation. Exosomes were characterized by NTA, transmission electron microscopy, and Western Blot; the RNA in EVs was extracted, and the library was built using the Hi-seq2500 sequencing platform for deep sequencing of the whole transcriptome (>30X); through genome comparison of tumor-related mutation sites, Analyze potential tumor driver genes and known drug targets.

[0023] 1 Ultra-high-speed centrifugation and purification: Centrifuge plasma at 3000g at low speed for 15 minutes to remove dead cells and large cell debris; filter the cell supernatant wi...

Embodiment 2

[0029] 1) Collect currently known tumor-related driver gene mutations and drug target mutations from oncoKB, IntoGen, and Cosmic. 2) Based on our previous experimental results, we screened out the gene pathways that can be stably detected in the body fluid exosomal RNA group and that are upregulated in metastatic osteosarcoma. 3) Finally, a gene combination including 158 genes was designed according to the intersection of the two. According to this combination, the blood exosomes of clinical patients can be extracted and purified, and after RNA extraction, the dynamic monitoring of osteosarcoma lung metastasis-related driver genes and drug target genes can be performed.

[0030] Drug target genes: ABL1, AKT1, ALK, AR, ATM, BRAF, BRCA1, BRCA2, CCND2, CDK4, CDK6, CDKN2A, CHEK2, CTNNB1, DNMT3A, EGFR, ERBB2, ERBB3, ERCC2, ESR1, FBXW7, FGF3, FGF4, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, LRP1B, MAP2K1, MDM2, MET, MYD88, NF1, NF2, NOTCH1, NOTCH2, N...

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Abstract

The invention relates to a detection method for metastatic osteosarcoma molecular targets, a gene combination and a kit. The method has the advantages as follows: the research firstly finds that 1) for the metastatic osteosarcoma, the extracellular vesica based whole transcriptome sequencing finds a molecular characteristic spectrum obviously related to metastasis and has the advantages of noninvasive property, dynamic tracking, and the like, and 2) abundant tumor bio-markers are gathered in the extracellular vesica of metastatic osteosarcoma, and the method has a significance in clinically assessing tumor metastasis and detecting drug targets. The invention designs the gene combination containing 158 genes. According to the gene combination, the driving genes and target genes related to the osteosarcoma pulmonary metastasis can be dynamically monitored after the purification for the blood exosome of the clinic patient and the RNA extraction.

Description

technical field [0001] The invention relates to the technical field of clinical medicine, in particular to the body fluid detection of molecular targets for the diagnosis and treatment of lung metastasis of osteosarcoma. Background technique [0002] The survival rate of osteosarcoma with lung metastasis is less than 20%, and the prognosis is extremely poor. However, due to the difficulty in early detection of lung metastasis of osteosarcoma, the difficulty in obtaining biopsy materials, and the heterogeneity of lung metastases, there is currently no effective method for the detection of molecular targets of metastatic osteosarcoma. This study intends to explore the possibility of detecting gene mutations and molecular targets related to lung metastasis of osteosarcoma based on extravesicle (EV) transcriptome sequencing through in vitro and in vivo experiments. [0003] Exosomes are vesicle bodies secreted by a variety of living cells, which contain various key components s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 鲍其远沈宇辉张伟滨
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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