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Polynucleotide for tumor treatment and application of polynucleotide

A polynucleotide and tumor treatment technology, applied to polynucleotides for tumor treatment and its application fields, can solve the problem of less research on lncRNA, and achieve the effect of inhibiting invasion and metastasis, and inhibiting the metastasis of gastric cancer.

Active Publication Date: 2019-03-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although lncRNA plays an important role in tumors, there are relatively few studies on lncRNA in gastric cancer

Method used

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  • Polynucleotide for tumor treatment and application of polynucleotide
  • Polynucleotide for tumor treatment and application of polynucleotide
  • Polynucleotide for tumor treatment and application of polynucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: LncRNA GMAN-AS is an lncRNA with a length of 2311nt

[0036] In order to detect the 5' and 3' end sequences of GMAN-AS, we based on SMARTer TM RACE cDNA Amplification Kit (purchased from Clontech, USA) was used for the experiment.

[0037] 1. According to the principle of designing primers in the Race manual, design the specific primers for Race as follows:

[0038] 5'race for GMAN-AS:5'-CTAGGAGTAGTATTAAGTGG-3'

[0039] 3'race for GMAN-AS:5'-CCCTGACTCACCGAAACAGCGT-3'

[0040] 2. Extract the RNA of human gastric cancer cell line and add PolyA treatment and purification to the RNA

[0041] 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. Gastric cancer cells HGC-27 (about 1×10 6 ) into 1mL TRIZOL, blow and pipe repeatedly 30 times with the pipette tip and transfer to 1.5ml RNase free EP tube, and let stand at room ...

Embodiment 2

[0090] Embodiment 2: GMAN-AS is expressed in human gastric tissue and gastric cancer cell lines

[0091] 1. Specific RNA probes for in vitro transcription of GMAN-AS

[0092] 1.1 Through NCBI's Blast tool, find a specific sequence of GMAN-AS, and design primers, and design GAPDH primers (purchased from Shanghai Sangong), as follows:

[0093]

[0094] A specific sequence of GMAN-AS / GAPDH was obtained by PCR reaction, the cDNA of human gastric cancer cell line HGC-27 was used as a template, and the above primers were used to carry out PCR reaction to obtain the target fragment.

[0095] 1.2 cDNA synthesis

[0096] The RNA of human gastric cancer cell lines was extracted and its concentration was measured according to TRIZOL reagent and instructions.

[0097] Take a PCR tube A and add 800ng of total RNA, make up DEPC water to a total volume of 10μL, and mix well. Take another PCR tube B and add 2 μL of 10×RT buffer, 2 μL of 10× random primer, 0.8 μL of dNTP, 1 μL of RNA rev...

Embodiment 3

[0156] Example 3: GMAN-AS can significantly inhibit the invasion of gastric cancer cells

[0157] 1. Cell transfection with siRNA

[0158] Two siRNAs specifically targeting GMAN-AS and silencing GMAN-AS expression were designed and synthesized by siRNA design software (purchased from Shanghai Gemma, China). Take the cells in the logarithmic growth phase and plate them. When the cell density reaches about 50%, transfect the system according to the following table, and dilute Lipo RNAi MAX (purchased from U.S. Invitrogen) and siRNA with OPTI medium (purchased from U.S. Gibco) respectively. Add the diluted siRNA to the Lipo RNAi MAX tube, mix well and let it stand for 5 minutes, then add it to the cell culture medium, shake well, and replace the medium after 24 hours.

[0159]

[0160] 2. Cell Transfection with Plasmids

[0161] 2.1 Construct the plasmid of pcDNA3.1-GMAN-AS

[0162] According to the results of Race, the full-length sequence of GMAN-AS was obtained, the prim...

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Abstract

The invention discloses a polynucleotide for tumor treatment. The polynucleotide comprises an MR fragment shown in SEQ ID NO.1 or further comprises an AMFR fragment shown in SEQ ID NO.2, wherein, theMR segment is a 565 bp nucleotide sequence, and the AMFR segment is a 293 bp nucleotide sequence. Both chemical synthesis and targeted gene editing technologies have the characteristics of simple operation and strong feasibility. The individual MR fragment, or AMFR fragment, and GMAN-AS containing the MR fragment and the AMFR fragment can significantly inhibit the invasion of gastric cancer cells,so that the polynucleotide has important potential for treating gastric cancer metastasis.

Description

technical field [0001] The present invention relates to the application of long-chain non-coding RNA GMAN-AS in tumor invasion and metastasis Background technique [0002] The rapid development of science and technology has also brought a profound impact on human life. Due to the rapid development of science and technology, the microchip technology and high-throughput sequencing technology for detecting the whole genome and transcriptome have been continuously improved, which has also brought great convenience to the research of biotechnology. The latest research shows that less than 2% of the genome has the ability to encode proteins, and at least 75% of the genome is transcribed as non-coding RNA. Although some non-coding transcripts are small non-coding RNAs, most of the transcripts are noncoding RNAs with a length of more than 200 nucleotides, so they are named long noncoding RNAs (long noncoding RNAs). , lncRNA). Studies have found that lncRNA is precisely regulated ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P35/00A61P35/04
CPCA61K31/713A61P35/00A61P35/04C12N15/1135C12N2310/10
Inventor 周天华卓巍刘易曼
Owner ZHEJIANG UNIV
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