RAB22A-NoeFs fusion gene system for diagnosing and/or treating osteosarcoma, and application thereof

A technology of rab22a-noefs and rab22a-noef1, applied in the field of RAB22A-NoeFs fusion gene line, can solve the problems of inability to evaluate clinical value and significance, achieve the effect of inhibiting migration and invasion, inhibiting lung metastasis, and promoting lung metastasis

Active Publication Date: 2018-12-07
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Frequent fusion genes reported in osteosarcoma include LRP1-SNRNP25, KCNMB4-CCND3 and PMP22-ELOVL5, among which LRP1-SNRNP25 and KCNMB4-CCND3 are related to the migration

Method used

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  • RAB22A-NoeFs fusion gene system for diagnosing and/or treating osteosarcoma, and application thereof
  • RAB22A-NoeFs fusion gene system for diagnosing and/or treating osteosarcoma, and application thereof
  • RAB22A-NoeFs fusion gene system for diagnosing and/or treating osteosarcoma, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Identification of RAB22A-NoeFs fusion gene sequence

[0040] Using RNA-seq, the RAB22A-NoeFs fusion gene was found in the human osteosarcoma cell lines ZOS and ZOSM. Subsequently, the full length of the CDS region of the RAB22A-NoeFs fusion gene was amplified to verify the obtained RAB22A-NoeFs fusion gene. The specific process is as follows:

[0041] 1. High-throughput sequencing identification of fusion genes in osteosarcoma cells:

[0042] Select primary osteosarcoma cells ZOS, osteosarcoma cell line ZOSM, and normal bone cells Hfob1.19, extract total RNA with TRIZOL, and send to Beijing Nuohezhiyuan Bioinformation Technology Co., Ltd. for library building and high-throughput second-generation transcriptome After sequencing and bioinformatics analysis, the RAB22A-NoeFs fusion gene was specifically expressed in the primary osteosarcoma cells ZOS and ZOSM (Table 1); the schematic diagram of the fusion method of the RAB22A-NoeFs fusion gene is shown in figu...

Embodiment 2

[0058] Example 2 Detection of RAB22A-NoeFs fusion gene

[0059] 1. RT-PCR detection

[0060]Extract the RNA of osteosarcoma cells ZOS, ZOSM and normal osteoblast Hhob1.19, reverse transcribe into cDNA; use the cDNA as a template, perform PCR verification on the RAB22A-NoeFs fusion gene, and perform Sanger sequencing on the PCR product; including the following steps:

[0061] (1) Design and screen specific RT-PCR primers, specific RT-PCR primers are as follows:

[0062] RAB22A-NoeFs-F: GGCCTCTCCCCTTCTCAACTTAG (SEQ ID NO: 13)

[0063] RAB22A-NoeF1-R: GTGGCTTTCTCAGCCGAAAC (SEQ ID NO: 14)

[0064] RAB22A-NoeF2-R: TCATTGATAGGCATCTGGGTTGGTT (SEQ ID NO: 15)

[0065] RAB22A-NoeF3-R: TCAGCCTCTAAGGTAGCTAGGACAA (SEQ ID NO: 16)

[0066] RAB22A-NoeF4-R: TCACCTGAGGTCAGGAGTTCGAGAC (SEQ ID NO: 17)

[0067] RAB22A-NoeF5-R: TTAGGTCATGTGCCCATATCTGAAC (SEQ ID NO: 18)

[0068] RAB22A-NoeF6-R: CTAAATCGATATTCCAATCTGGTCT (SEQ ID NO: 19)

[0069] (2) Trizol method to extract RNA

[0070] Add 1...

Embodiment 3

[0093] Example 3 Function of RAB22A-NoeFs fusion gene in osteosarcoma

[0094] 1. Construction of stable overexpression cells

[0095] 1. Construction of overexpression vector

[0096] (1) CDS region sequence amplification of RAB22A-NoeFs

[0097] Using TAKARA's high-fidelity enzyme MIX primSTAR, the specific primers in Example 2 were used to amplify the CDS region sequence of RAB22A-NoeFs. The system is as follows:

[0098]

[0099] Place it in a PCR instrument after mixing it evenly, and the reaction conditions are as follows:

[0100] After pre-denaturation at 95°C for 5 minutes, carry out 38 cycles under the following conditions:

[0101] 98°C 15s

[0102] 55-60℃ 30s

[0103] 72℃ 5kb / min

[0104] After 38 cycles, a further step of 72°C, 10min extension reaction was performed, after which the amplified product was recovered;

[0105] (2) Digestion (including overexpression vector PBABE and amplification products)

[0106] Perform digestion reactions in 200 μL PCR...

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Abstract

The invention discloses a fusion gene line RAB22A-NoeFs for diagnosing and/or treating osteosarcoma. The first and second exons of the RAB22A gene are fused with the 54408036th to 54476718th positionof chromosome 20 after being inversed, the fusion gene line includes the following six fusion genes: RAB22A-NoeF1, RAB22A-NoeF2, RAB22A-NoeF3, RAB22A-NoeF4, RAB22A-NoeF5 and RAB22A-NoeF6, and the nucleotide sequences of the fusion genes are sequentially represented by SEQ ID NO:1-6. The six fusion genes of the fusion gene line RAB22A-NoeFs can promote the migration and invasion of osteosarcoma cells, wherein the RAB22A-NoeF1 has above functions, and also can promote the lung metastasis of the osteosarcoma cells. The fusion gene line RAB22A-NoeFs can be used as a specific therapeutic target forosteosarcoma, and has a great application prospect.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically, relates to a RAB22A-NoeFs fusion gene line for diagnosing and / or treating osteosarcoma and its application. Background technique [0002] Osteosarcoma (Osteosarcoma) is a malignant bone tumor that occurs frequently in children and adolescents. It is prone to lung metastasis. It is highly malignant and harmful. Its 5-year survival rate is only about 60%. The main treatment method is neoadjuvant chemotherapy + surgery + Chemotherapy after surgery, and it is very insensitive to chemoradiotherapy, which seriously affects the quality of life of patients and there is no effective targeted drug at present. Surgical treatment usually uses amputation, so it is very important to find a new way to treat osteosarcoma. Urgent and necessary. [0003] Whether there is a specific therapeutic target in osteosarcoma is the fundamental issue in the treatment of osteosarcoma. Understandin...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/82C12Q1/6886C12N15/11A61K45/00A61K31/7088A61P35/00
CPCA61K31/7088A61K45/00A61P35/00C07K14/82C12Q1/6886
Inventor 康铁邦廖丹钟理沈靖南隋建华
Owner SUN YAT SEN UNIV CANCER CENT
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