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miRNA diagnostic markers for osteosarcoma

A technology for osteosarcoma and sarcoma, applied in the field of miRNA diagnostic markers

Active Publication Date: 2019-08-20
杨祚璋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the differential expression of miRNAs in osteosarcoma. Finding and identifying miRNAs related to the occurrence of osteosarcoma provides the basis for clinical diagnosis and treatment of miRNAs, and helps the early diagnosis and prognosis evaluation of osteosarcoma to reach a new level.

Method used

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  • miRNA diagnostic markers for osteosarcoma
  • miRNA diagnostic markers for osteosarcoma
  • miRNA diagnostic markers for osteosarcoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Screening of miRNAs associated with osteosarcoma

[0043] 1. Sample acquisition: 10 patients with chondroblastic osteosarcoma and 10 normal people were collected. Subjects were required to fast for at least 12 hours, and at room temperature from 7:00 to 8:00 in the morning of the next day, 10ml of venous blood was drawn into EDTA anticoagulant tubes to extract PBMCs from peripheral blood mononuclear cells, and 1ml of Trizol was added Reagents (Invitrogen Company), mixed thoroughly, and samples were stored 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.

[0044] 2. Extraction of blood mononuclear cell RNA

[0045] Total RNA was extracted using the RNA extraction kit from Invitrogen. Proceed as follows:

[0046] (1) First add 1ml Trizol to 1×10 7 In the cells, if the frozen cells are directly added to Trizol, ther...

Embodiment 2

[0062] Example 2 QPCR verification of differentially expressed miRNA-4521

[0063] 1. Select miRNA-4521 according to the detection results of the miRNA chip for large sample QPCR verification. According to the sample collection method in Example 1, 60 samples of normal people and 60 samples of chondroblastic osteosarcoma were selected.

[0064] 2. The RNA extraction process is the same as in Example 1.

[0065] 3. Reverse transcription:

[0066] 1) Mix 10pg-1μg total RNA template with 2μl 10× buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl RNase inhibitor and RNase free water , the final volume was 20 μl, and incubated at 37°C for 1h.

[0067] 2) Add 1 μl of 0.5 μg / μl Oligo(dT) specific RT primer to the reaction tube, and incubate at 70° C. for 5 minutes.

[0068] 3) Immediately incubate on ice for at least 2 minutes to break the secondary structure of RNA and primers.

[0069] 4) Mix the above 20 μl reaction mixture with 4 μl 5× buffer, 1 μl dNTP (10 mM), 0.5 μl M...

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Abstract

The invention discloses an miRNA diagnosis marker of osteosarcoma. The miRNA marker is miRNA-4521. On the basis of high-throughput sequencing, it discovers that the expression of the miRNA-4521 in the blood of a patient with the osteosarcoma is down-regulated; and by conducting large-sample detection by virtue of qPCR, it further proves that the miRNA-4521 is related to the occurrence and development of the osteosarcoma. Therefore, the invention provides a means and a product for achieving the early diagnosis of the osteosarcoma, so that the early interventional therapy of the patient with the osteosarcoma is achieved, and the survival rate and the survival quality of the patient with the osteosarcoma are improved.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a miRNA diagnostic marker for osteosarcoma, specifically the miRNA diagnostic marker is miRNA-4521. Background technique [0002] Osteosarcoma (Osteosarcoma) is a malignant tumor originating from the bone, and its incidence rate ranks first among primary bone malignant tumors. Osteosarcoma is divided into various types. As a subtype of common osteosarcoma, chondroblastic osteosarcoma has no significant difference in clinical treatment and prognosis from other common osteosarcomas. 75% of osteosarcoma patients are teenagers, more males (male:female 1.5:1), the degree of malignancy is very high, early lung metastasis is prone to occur, and the prognosis is extremely poor. Once found in the past, amputation is often used for treatment. Even so, patients The postoperative 5-year survival rate is only 5-20%, which brings great mental harm to patients and their families, and also brings heavy ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
Inventor 杨祚璋杨义豪李东奇张娅
Owner 杨祚璋