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New application of mir-1299 and mature miRNA thereof

1. mir-1299, a mature technology, applied in the field of molecular biology, can solve the problem of lack of effective targets for the diagnosis and treatment of osteosarcoma

Active Publication Date: 2016-04-13
GUAN BOJIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment at the gene level has become a research hotspot for many scholars, but there is still a lack of effective targets for the diagnosis and treatment of osteosarcoma. Further search for molecular biomarkers will have important theoretical and clinical application value
At the same time, if the serum diagnostic markers of osteosarcoma can be obtained, it will have a profound impact on the diagnosis and treatment of osteosarcoma

Method used

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  • New application of mir-1299 and mature miRNA thereof
  • New application of mir-1299 and mature miRNA thereof
  • New application of mir-1299 and mature miRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The collection of embodiment 1 sample and total RNA extraction

[0061] 5 patients with metastatic osteosarcoma and 5 healthy controls. Five patients in the metastatic osteosarcoma group and five in the control group were required to fast for at least 12 hours, and at room temperature from 7:00 to 8:00 the next morning, 10ml of venous blood was drawn into ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and extracted Peripheral blood mononuclear cell PBMCs, add 1ml Trizol reagent (Invitrogen Company), mix thoroughly, 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.

[0062] RNA extraction standards: RNA purity: OD260 / 280≧1.8, 28S / 18S≧1; RNA integrity: RIN value≧7.0. RNA integrity detection method: Agilent2100 (RNA6000Nanokit), agarose gel electrophoresis (agarose gel concentration: 1% agarose...

Embodiment 2

[0063] Example 2 Sequencing and Data Analysis

[0064] Sequencing: mRNA and miRNA were sequenced using lluminaHiseq2500 / Miseq second-generation high-throughput sequencing technology, and data processing was completed through the processes of removing joints, removing low-quality, and decontaminating to obtain the final data.

[0065] The miRNA and mRNA 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 miRNA and mRNA. Setting p value <0.01, 12 differentially expressed miRNAs were screened out, including 11 miRNAs whose expression levels were down-regulated. During the analysis, the P value was set <0.05, and a total of 425 differentially expressed mRNAs were screened out, including 324 genes whose expression levels were upregulated. Use algorithms including RNA22, miRanda, miRDB, miRWalk, PICTAR2 a...

Embodiment 3

[0066] Example 3Real-timePCR detection of miR-1299 and C5AR1 and ITPRIP gene expression in osteosarcoma tissue

[0067] 1 sample collection:

[0068] The peripheral blood of 49 patients with metastatic osteosarcoma tumors and 63 healthy controls came from the hospital (collection time from April 2014 to August 2015).

[0069] 2 total RNA extraction:

[0070] Removal of RNase for related experimental items:

[0071] ① Rinse and soak all glassware with DEPC before use, 120°C for 20 minutes under high pressure, and 180°C for more than 2 hours.

[0072] ②Plastic utensils (such as: EP tubes / tips) need to be soaked in 0.1% DEPC water overnight before use, and then the liquid is controlled to dry, 120°C and high pressure for 20 minutes, and dried in an oven for later use.

[0073] leukapheresis

[0074] (1) Take 2m1 anticoagulated peripheral blood (blood collection time does not exceed 3h);

[0075] (2) Add an equal volume of sterile PBS to the peripheral blood and mix thoroughl...

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Abstract

The invention relates to new application of mir-1299 and mature miRNA thereof. According to the new application, patients with metastatic osteosarcoma and a healthy contrast crowd are sequenced to obtain expression data of miRNA and mRNA thereof; candidate miR-1299 genes and target genes C5AR1 and ITPRIP of the miR-1299 genes are screened out; further, a fluorescent quantitative experimental result shows that the miR-1299 genes and the target genes thereof are closely related to osteosarcoma, and migration and invasion of osteosarcoma cells can be effectively inhibited by inhibiting expression of the miR-1299 genes. The new application provides a good diagnosis and treatment target spot for osteosarcoma metastasis clinically and has high practical application value.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to the new application of mir-1299 and its mature miRNA, and more specifically to the new application of mir-1299 and its mature miRNA in the diagnosis and treatment of osteosarcoma metastasis. Background technique [0002] miRNA is an endogenous non-coding small molecule that regulates gene expression by specifically binding to target genes. There are two main ways of its regulation: one is incomplete complementary pairing with the 3'UTR of the target gene mRNA, blocking the translation of the target gene, thereby regulating gene expression; the other is similar to siRNA, when miRNA and mRNA are completely When complementary pairing, Ago2 protein cuts mRNA and directly leads to its degradation, realizing gene silencing. In conclusion, it is currently believed that the way miRNAs interact with target genes and the degree of pairing between miRNAs and target genes are relate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 李曙光杜玉梅杨承刚
Owner GUAN BOJIAN BIOTECH CO LTD