Tumor-associated sequence and long non-coding RNA and application thereof

A long-chain non-coding, tumor-related technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of low survival rate, difficult surgical resection and poor prognosis of patients

Active Publication Date: 2019-04-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the onset of gastric cancer organ metastases is hidden, and most patients are difficult to be surgically removed when discovered, so the prognosis is extremely poor, and its 5-year survival rate is less than 10%, which is one of the main causes of death in patients with advanced gastric cancer

Method used

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  • Tumor-associated sequence and long non-coding RNA and application thereof
  • Tumor-associated sequence and long non-coding RNA and application thereof
  • Tumor-associated sequence and long non-coding RNA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: lncRNA GMAN is an 855nt lncRNA

[0067] In order to detect the 5' and 3' end sequences of GMAN, we based on SMARTer TM RACE cDNA Amplification Kit (purchased from Clontech, USA) was used for the experiment.

[0068] 1. According to the principle of designing primers in the Race manual, design the specific primers for Race as follows:

[0069] 5'race for GMAN:5'-CAAGACTTCTATACCAT-3'

[0070] 3'race for GMAN:5'-CAGGCTGGTCTCGAACTCCT-3'

[0071] 2. Extraction of RNA from human gastric cancer cell lines and processing and purification of RNA with PolyA

[0072] The isolation and extraction of total cellular RNA was performed using TRIZOL reagent (purchased from Invitrogen, USA) and performed according to the standard operation of the reagent manual. An appropriate amount of RNA was used to digest the genome remaining in the RNA using a DNase I kit (purchased from Invitrogen, USA) to remove genome contamination.

[0073] RNA was treated with PolyA by using Po...

Embodiment 2

[0107] Example 2: GMAN is expressed in human gastric tissue and gastric cancer cell lines

[0108] 1. Specific RNA probes for in vitro transcription of GMAN (targeting the MFR region)

[0109] 1.1 Through the Blast tool of NCBI, find a specific sequence of GMAN, and design primers, and design the primers of GAPDH (purchased from Shanghai Sangong), as follows:

[0110]

[0111] A specific sequence of GMAN / GAPDH was obtained by PCR reaction, and the cDNA of human gastric cancer cell line was used as a template, and the above primers were used to carry out PCR reaction to obtain the target gene (purchased from Toyobo, Japan). Take a PCR tube, add 2ul of cDNA template, 1ul of KOD high-fidelity enzyme, 5ul of 10xPCR buffer, 5ul of dNTP, 1ul of Forward primer, 1ul of reverse primer, add ddH2O to 50ul, mix well, and perform PCR reaction according to the following reaction procedure:

[0112] 95℃

2min

95℃

30s

Tm-5℃

30s

68℃

1min-2min (extens...

Embodiment 3

[0128] Example 3: GMAN is relatively highly expressed in gastric cancer tissue and is significantly correlated with metastasis and poor prognosis of gastric cancer patients

[0129] 1. Reverse transcription of RNA from gastric cancer tissue and its paired normal tissue into cDNA

[0130] The tissue was ground into a powder with a mortar at an extremely low temperature, and the total RNA of the powdered tissue was isolated and extracted using TRIZOL reagent (purchased from Invitrogen, USA) and performed according to the standard operation of the reagent manual. The extracted RNA was subjected to reverse transcription PCR to synthesize cDNA products.

[0131] 2. Real-time fluorescent quantitative PCR (QRT-PCR) detection of the expression of GMAN in gastric cancer tissue and its paired normal tissue

[0132] The PCR reaction system was prepared according to the instructions of Taqman probe Kit (purchased from Takara, Japan). The fluorescent quantitative PCR reaction was carried...

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Abstract

The invention discloses a tumor-associated sequence, a tumor-associated long non-coding RNA (lncRNA) and application of the tumor-associated sequence. lncRNA GMAN (gastric cancer metastasis associatedlong noncoding RNA) has relatively high expression in gastric cancer and particularly has remarkably high expression in samples of patients with M1-stage gastric cancer generating distant metastases,and the high expression of the GMAN is remarkably related to poor prognosis of the patients with the gastric cancer. A research shows that the GMAN plays an important role in occurrence and development of the gastric cancer; the GMAN can be used for remarkably promoting invasion and metastasis of gastric cancer cells. A therapeutic experiment of CRISPR / Cas9 shows that the GMAN, in particular to an MFR (mannosylfucosyl receptor) segment of the GMAN, can be used for effectively inhibiting the metastasis of the gastric cancer and can serve as a novel strategy for controlling the metastasis of tumors clinically.

Description

technical field [0001] The invention relates to a tumor-associated sequence, long-chain non-coding RNA and applications thereof. Background technique [0002] Recent transcriptome studies have shown that more than 70% of the human genome is transcribed as RNA, most of which are non-coding transcripts. There are at least 20 known non-coding transcripts, which are generally divided into two categories, one is small non-coding RNA (sncRNA, Small non-coding RNA), and its transcript length is less than 200 nucleotides, and the other is The category is long non-coding RNA (Long non-coding RNA, LncRNA), which is broadly defined as RNA with a transcription length of more than 200 nucleotides and no apparent coding ability. According to the position of LncRNA gene, it can be divided into five categories: sense LncRNA, antisense LncRNA, bidirectional LncRNA, intron LncRNA and intergenic LncRNA. At present, more and more evidence shows that many non-coding RNAs have important biologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/113
CPCC12N15/113C12N2310/10C12Q1/6886C12Q2600/118C12Q2600/178
Inventor 周天华卓巍刘易曼
Owner ZHEJIANG UNIV
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