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Application of EB virus encoded microRNA BART6-3p to preparation of nasopharyngeal cancer prognostic preparation

A technology for Epstein-Barr virus and nasopharyngeal carcinoma, applied in the field of tumor molecular biology, has achieved important promotion and application prospects and far-reaching clinical significance

Inactive Publication Date: 2016-01-13
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previously, there was no literature report on the function of LOC553103

Method used

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  • Application of EB virus encoded microRNA BART6-3p to preparation of nasopharyngeal cancer prognostic preparation
  • Application of EB virus encoded microRNA BART6-3p to preparation of nasopharyngeal cancer prognostic preparation
  • Application of EB virus encoded microRNA BART6-3p to preparation of nasopharyngeal cancer prognostic preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1, real-time fluorescent quantitative PCR method detection confirmed that BART6-3p was up-regulated in nasopharyngeal carcinoma

[0075] 1. Materials and methods:

[0076] Collect 5 cases of normal nasopharyngeal epithelial tissues and 18 cases of nasopharyngeal carcinoma tissues, extract total RNA with Trizol (product of Invitrogen Company), reverse transcribe 2 μg RNA into cDNA with miScript reverse transcription kit (product of Qiagen Company), and use QuantiTectSYBRGreenPCR kit (Qiagen company product) Real-time fluorescent quantitative PCR was used to detect the expression of BART6-3p and internal reference gene RNU6B. The public primers (UniversalPrimer) of microRNA and the specific primers of BART6-3p and RNU6B were all designed and synthesized by Qiagen.

[0077] Real-time fluorescence quantitative PCR reaction system

[0078]

[0079] Real-time fluorescent quantitative PCR reaction steps

[0080] 194℃5min

[0081] 295℃10sec

[0082] 358℃30sec

...

Embodiment 2

[0092] Example 2, in situ hybridization detection found that the expression of BART6-3p in nasopharyngeal carcinoma and gastric carcinoma is related to the prognosis of patients

[0093] 1. Material method

[0094] 1.1 Design and synthesis of hybridization probes

[0095] In order to detect the expression of BART6-3p by in situ hybridization, we designed an oligonucleotide probe for detecting the expression of BART6-3p by in situ hybridization and a positive control in situ hybridization oligonucleotide probe.

[0096] BART6-3p probe: UCUAAGGCUAGUCCGAUCCCCG

[0097] Positive control probe (to detect the housekeeping gene GAPDH):

[0098] GAPDH probe: CAGUAGAGGCAGGGAUGAUGUUCU

[0099] The gene-specific oligonucleotide probe sequences designed above were synthesized by chemical synthesis method, and uracil in the probe sequences was labeled with biotin (bio-U) during the synthesis process.

[0100] 1.2 Oligonucleotide probe labeling kit and in situ hybridization detection re...

Embodiment 3

[0166] Example 3, Overexpression of BART6-3p inhibits growth, proliferation, invasion and metastasis of tumor cells

[0167] 1. Material method

[0168] 1.1 Cell culture and transfection

[0169] EBV-negative nasopharyngeal carcinoma cell lines 5-8F, HNE2, and gastric cancer cell AGS were purchased from the Cell Center of Central South University. The RPMI1640 medium and fetal bovine serum used for cell culture, and the trypsin used for digesting cells were all products of Gibco, USA.

[0170] BART6-3p is synthesized by Invitrogen Company through chemical synthesis, and the sequence is:

[0171] CGGGGAUCGGACUAGCCUUAGA

[0172] Tumor cell lines with good growth status were divided into 2×10 5 Cells / well were seeded in a 6-well plate, and the 6-well plate was placed at 37°C, 5% CO 2 In the incubator, the transfection of BART6-3p can be started when the cells to be cultured grow to a density of 50-70%; the transfection process is as follows:

[0173] Add 8 μl of Hiperfect tr...

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Abstract

The invention discloses application of EB virus encoded microRNA BART6-3p to preparation of a nasopharyngeal cancer prognostic preparation. EB virus encoded microRNA BART6-3p is mainly used for preparing preparations for monitoring recurrence and metastasis and forecasting prognosis of nasopharyngeal cancer. Close relation between expression of BART 6-3p and prognosis of a nasopharyngeal cancer patient is discovered in a filed nasopharyngeal cancer paraffin-embedded tissue sample through in-situ hybridization, negative correlation between the expression level of BART6-3p and metastasis of the nasopharyngeal cancer patient is detected in nasopharyngeal cancer tissues, the lifetime of the patient with high expression of BART6-3p is longer, and the BART6-3p can serve as a predictive molecular marker of recurrence and metastasis of nasopharyngeal cancer. The application provided by the invention provides a powerful molecular biology tool for auxiliary diagnosis and prognosis forecasting of nasopharyngeal cancer and has profound clinical significance and important popularization and application prospects.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and specifically relates to a method for preparing preparations for monitoring recurrence and metastasis of nasopharyngeal carcinoma and predicting prognosis by using microRNABART6-3p encoded by Epstein-Barr virus. Background technique [0002] Epstein-Barrvirus (EBV) is a ubiquitous human herpes virus that can infect about 95% of the adult population in the world. Except for a small number of people who occasionally cause infectious mononucleosis, most of the infected people There will not be any clinical symptoms, and EBV can remain latent in the host for a long time in most cases. However, latently infected EBV can lead to malignant tumors in some cases, including B-cell-derived Burkitt and Hodgkin's lymphoma, and epithelial-derived nasopharyngeal and gastric carcinomas. The mechanism of EBV leading to malignant tumors is not yet fully understood. It may be related to the expression of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6886C12Q1/705C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 曾朝阳何宝玉熊炜李桂源李小玲李夏雨张文玲石磊范松青龚朝建周鸣周艳宏
Owner CENT SOUTH UNIV
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