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A kind of rab22a-noefs fusion gene line and application thereof for diagnosis and/or treatment of osteosarcoma

A technology of rab22a-noefs and rab22a-noef1, which is applied in the field of RAB22A-NoeFs fusion gene line, can solve the problems that the clinical value and significance cannot be evaluated, and achieve the effects of inhibiting migration and invasion, promoting lung metastasis, and inhibiting lung metastasis

Active Publication Date: 2021-09-07
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 of osteosarcoma cells, but unfortunately, in these positive cases , whose protein product has not been detected, so its clinical value and significance cannot be assessed

Method used

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  • A kind of rab22a-noefs fusion gene line and application thereof for diagnosis and/or treatment of osteosarcoma
  • A kind of rab22a-noefs fusion gene line and application thereof for diagnosis and/or treatment of osteosarcoma
  • A kind of rab22a-noefs fusion gene line and application thereof for diagnosis and/or treatment of osteosarcoma

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 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 tubes. ...

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Abstract

The invention discloses a fusion gene line RAB22A‑NoeFs for diagnosing and / or treating osteosarcoma, in which exons 1 to 2 of the RAB22A gene are inverted and fused with chromosome 20 at 54408036‑54476718, comprising 6 Fusion gene: RAB22A-NoeF1, RAB22A-NoeF2, RAB22A-NoeF3, RAB22A-NoeF4, RAB22A-NoeF5 or RAB22A-NoeF6, the nucleotide sequences of which are shown in SEQ ID NO: 1-6. The six fusion genes of the fusion gene line RAB22A‑NoeFs can all promote the migration and invasion of osteosarcoma cells, among which RAB22A‑NoeF1 can also promote the lung metastasis of osteosarcoma cells in addition to the above functions. The fusion gene line RAB22A-NoeFs of the present invention can be used as a specific therapeutic target for osteosarcoma, and has great application prospects.

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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Patent Type & Authority Patents(China)
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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